Reagent cartridge and methods for detection of cells

ABSTRACT

An apparatus includes a housing and an actuator. The housing, which defines a reagent volume that can receive a reagent container, can be removably coupled to a reaction chamber. The housing includes a puncturer that defines a transfer pathway in fluid communication with the reagent volume. A delivery portion of the housing defines a delivery pathway between the transfer pathway and the reaction chamber when the housing is coupled to the reaction chamber. The actuator has a plunger portion disposed within the reagent volume. An engagement portion of the actuator can be manipulated to move the plunger portion within the reagent volume to deform the reagent container. The puncturer can pierce a frangible portion of the reagent container to convey a reagent from the reagent container into the reaction chamber via the transfer pathway and/or the delivery pathway.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/617,631, entitled “Reagent Cartridge and Methods for Detection ofCells,” filed Feb. 9, 2015, which is a continuation-in-part of U.S.patent application Ser. No. 14/476,392, now abandoned, entitled “ReagentCartridge and Methods for Detection of Cells,” filed Sep. 3, 2014, whichclaims priority to and the benefit of U.S. Provisional PatentApplication Ser. Nos. 61/983,765, entitled “Reagent Cartridge forDetection of Cells,” filed Apr. 24, 2014; 61/939,126, entitled “Systemsand Methods for Packaging Nucleic Acid Molecules into Non-ReplicativeTransduction Particles and Their Use as Cellular Reporters,” filed Feb.12, 2014; and 61/897,040, entitled “Transcript Detection Systems andMethods,” filed Oct. 29, 2013, each of which is incorporated herein byreference in its entirety.

U.S. patent application Ser. No. 14/617,631 also claims priority to andthe benefit of U.S. Provisional Patent Application Ser. Nos. 61/983,765,entitled “Reagent Cartridge for Detection of Cells,” filed Apr. 24,2014; and 61/939,126, entitled “Systems and Methods for PackagingNucleic Acid Molecules into Non-Replicative Transduction Particles andTheir Use as Cellular Reporters,” filed Feb. 12, 2014, each of which isincorporated herein by reference in its entirety.

BACKGROUND

The embodiments described herein relate to systems and methods fordetection of cells using engineered transduction particles. Moreparticularly, the embodiments described herein also relate to acontainer and instrument within which the detection of bacteria can beperformed in an integrated, closed system with walkaway functionality.

Detection of bacteria, especially drug resistant strains, is a criticalstep in diagnosing and limiting spread of bacterial infections. Forexample, MRSA is a drug-resistant version of the common Staphylococcusaureus bacteria that is carried by a significant portion of thepopulation in the U.S. Most infections of MRSA occur in hospitals, andcan have a high mortality rate (MRSA infections kill approximately19,000 people in the U.S. every year). Accordingly, there is a need forefficient, accurate and rapid identification of the bacterial strains(including their phenotype and/or genotype and other molecular targets)that cause infection, such as MRSA. Particularly important is theability to identify the bacterial phenotype and/or genotype and othermolecular targets from a variety of different samples (e.g., humansamples, environmental samples, plant samples, veterinary samples, foodsamples or the like), so that the appropriate treatment and controlregimen can be started in a timely fashion.

One known method for identifying bacteria includes bacterial culture.Culturing is highly sensitive, but often takes 18 hours or more to yielda result, and is therefore not suitable for rapid diagnosis or forefficient screening purposes. Known culturing methods are oftenperformed using systems that require highly trained personnel to performthe assay, and are therefore not suitable for use in a variety ofdifferent settings. Known culturing methods are also prone tocontamination, which can result in false positives and/ormisidentification of the bacteria. Moreover, known culturing methodsemploy specifically tailored culture protocols for identification ofvarious bacterial species, thus testing a broad bacteria panel canrapidly elevate the cost.

Direct bacterial immunodetection, that is, detection using an antibodyantigen reaction, is another method for bacterial detection. Knownmethods of immunodetection can produce results more quickly and at alower cost than a culture, but are often limited by the availability ofselective antibodies for the bacterial strain of interest and availableantibodies are prone to cross-reactivity. Such known methods are alsoless sensitive than culturing, so there is often nevertheless arequirement of bacterial amplification that can lengthen the assay time.

Other known methods for detection of bacterial cells include isolationand analysis of nucleic acid such as DNA or RNA. Known methods forisolating nucleic acids from a sample often include several stringentsample preparation steps that require expensive and specializedequipment. In particular, such steps include 1) removing the proteinswithin a sample containing bacteria or cells by adding a protease; 2)breaking down the remaining bulk sample to expose the nucleic acidscontained therein (also referred to as cell lysing); 3) precipitatingthe nucleic acid from the sample; 4) washing and/or otherwise preparingthe nucleic acid for further analysis; 5) analyzing the nucleic acid toidentify the species. After preparing the sample, known analysis methodscan include polymerase chain reaction (PCR), gene sequencing, genefingerprinting, fluorescence, immunoassay, electrochemical immunoassay,microarrays, any other suitable technique or a combination thereof. PCRhas found widespread commercial usage but often requires multiple stepsinvolving expensive reagents and instrumentation. Many known methodsinvolving PCR are not suitable for bench top testing (e.g., they requirerelatively skilled personnel). Moreover, known PCR methods employthermal cycling and/or elevated temperatures, which can increase thecost, time and/or complexity of the analysis. In addition, becausenucleic acid amplification based techniques do not measure the responseof a bacteria to an antibiotic, such techniques are not suitable forantibiotic susceptibility testing. Finally, because nucleic acidamplification methods lyse the sample cells, such methods cannotdistinguish between live and dead cells.

Some known systems and methods for cell identification include the useof bacteriophages to identify and/or detect certain bacteria. In someknown methods, phages that are tagged with a reporter molecule can beused to target and infect a specific bacterial strain. After infection,the phages can undergo a lytic cycle (i.e., break the cell wall killingthe target bacteria) and/or a lysogenic cycle (i.e., replication of thephage along with the bacteria without killing the bacteria), followed bydetection of the amplified progeny phage. Such known methods relying onphage detection often include limiting or complex steps. For example,some known phage detection-based methods for identification rely onphage replication (during which the bacteria can be lysed), andtypically require cell culturing for facilitating this process. Someknown phage detection-based methods require removal or “unbinding” ofspecifically bound phages from the samples using carefully meteredand/or pH controlled reagents. Moreover, some known phagedetection-based methods rely on careful metering of the amount of phageadded and/or include opening or closing of the reaction chamber toadd/remove reagents, which can lead to contamination and/or prematuremixing of reagents leading to erroneous results and making the assaycomplex in nature.

Some known phage based systems and methods can result in undesirableand/or inconsistent delivery of reagents into a closed system. Forexample, some known systems and methods deliver reagents into a sampleto facilitate a reaction that can be optically detected. Inconsistentand/or inaccurate delivery of such reagents can result in undesirablevariability associated with the light detection, potentially falsereadings or the like. Some known systems employ sealed reagentcontainers or “blister packs” to isolate the reagents and the sampleuntil delivery of the reagents is desired. To facilitate delivery ofreagents from a blister pack, some known systems include mechanisms,such as rollers, to expel the reagent (see, e.g., WO2005/085855, FIG.31). Other known systems include multiple puncturers to facilitate therupture of a blister pack (see e.g., WO2007/115378, FIG. 16). Excessive“dead volume” (the volume within a blister pack after actuation that cancontain the reagent), however, can result in inconsistent delivery timesand/or amounts. Moreover, delivery mechanisms of known systems canproduce undesired effects when the reagent is delivered (e.g., excessivesplash or incomplete mixing). Thus, many known systems do notaccommodate delivery of reagents associated with a flash luminescencereaction.

In addition to the above-described drawbacks regarding the use ofphage-based methods, known methods do not employ automation orinstrumentation for enabling a “walk away” bacteriophage identificationsystem. For example, many known systems do not accommodate closed systemhandling and/or measurement of a signal that is produced by certainreporter molecules, such as for example, a flash luminescence reaction.Thus, known systems and methods require skilled personnel and intimatehandling of the samples, which can increase the possibility of falsepositives or negatives.

Thus, a need exists for improved apparatus and methods for rapid, costeffective and facile detection and identification of bacterial speciesin clinical samples. In particular, a need exists for improved rupturestructures and delivery paths within such systems. In addition, a needexists for improved apparatus and methods for efficient storage andtransfer of clinical samples from a point of collection to a testinglocation.

SUMMARY

Systems and methods for detecting and/or identifying target cells (e.g.,bacteria) using engineered vectors (including viral vectors) and/ortransduction particles are described herein. In some embodiments, anapparatus includes a housing and an actuator. The housing, which definesa reagent volume that can receive a reagent container, can be removablycoupled to a reaction chamber. The housing includes a puncturer thatdefines a transfer pathway in fluid communication with the reagentvolume. A delivery portion of the housing defines a delivery pathwaybetween the transfer pathway and the reaction chamber when the housingis coupled to the reaction chamber. The actuator has a plunger portiondisposed within the reagent volume. An engagement portion of theactuator can be manipulated to move the plunger portion within thereagent volume to deform the reagent container. The puncturer can piercea frangible portion of the reagent container to convey a reagent fromthe reagent container into the reaction chamber via the transfer pathwayand/or the delivery pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 are schematic illustrations of a container assembly accordingto an embodiment, in a first configuration, second configuration andthird configuration, respectively.

FIG. 4 is a cross-section view of a portion of the container assemblyshown in FIGS. 1-3 taken along the line X-X in FIG. 1.

FIG. 5 is a schematic illustration of a container assembly according toan embodiment.

FIGS. 6 and 7 are schematic illustrations of a container assemblyaccording to an embodiment, in a first configuration and secondconfiguration, respectively.

FIG. 8 is a top view of a portion of the container assembly shown inFIGS. 6 and 7.

FIG. 9 is a schematic illustration of a portion of the containerassembly shown in FIGS. 6 and 7 being deformed in response to an appliedforce.

FIGS. 10 and 11 show a perspective view and an exploded view,respectively, of a container assembly 4700, according to an embodiment.

FIG. 12 is a top perspective view of a housing of the container assemblyshown in FIGS. 10 and 11.

FIG. 13 is a cross-sectional view of the housing of the containerassembly shown in FIGS. 10 and 11.

FIG. 14 is an enlarged view of the portion of the housing identified asregion Z in FIG. 13.

FIG. 15 is a cross-sectional view of a reagent container of thecontainer assembly shown in FIGS. 10 and 11.

FIG. 16 is a cross-sectional view of an actuator of the containerassembly shown in FIGS. 10 and 11.

FIGS. 17 and 18 are cross-sectional views of the container assemblyshown in FIGS. 10 and 11 in a first configuration and a secondconfiguration, respectively.

FIG. 19 is an enlarged view of the portion of the container assemblyidentified as region Z in FIG. 18.

FIG. 20 is a kit, according to an embodiment, that includes thecontainer assembly shown in FIGS. 10 and 11.

FIG. 21 is a schematic flow diagram of a method according to anembodiment.

FIG. 22 is a flow chart of a method according to an embodiment.

FIG. 23 is a flow chart of a method according to an embodiment.

FIG. 24 is a cross-sectional view of a housing assembly according to anembodiment.

FIGS. 25A-25C are a side view, a front view and a bottom view,respectively, of a housing assembly according to an embodiment.

FIGS. 26 and 27 are front views of a housing assembly according to anembodiment

FIG. 28 is a schematic illustration of a test procedure, and the testresults of a comparison of Rayon wound swabs vs. Nylon foam swabs.

FIG. 29 is a schematic illustration of a test procedure comparing woundswabs, flock swabs and foam.

FIGS. 30A and 30B are bar charts showing the results from the testidentified in FIG. 29.

FIG. 31 is a bar graph of the results from a test comparing variousdifferent injection speeds of a substrate.

DETAILED DESCRIPTION

Systems and methods for detecting and/or identifying target cells (e.g.,bacteria) using engineered vectors (including viral vectors) and/ortransduction particles are described herein. In some embodiments, anapparatus includes a housing and an actuator. The housing, which definesa reagent volume that can receive a reagent container, can be removablycoupled to a reaction chamber (e.g., which can contain a sampleincluding a target cell). The reagent container can be disposed withinthe reagent volume, and can contain any suitable reagent or substance.For example, the reagent container can contain one or more transductionparticles, a reagent formulated to react with one or more reportermolecules in a sample to enhance production of a signal or otherwiseaugment the signal or other assay components, tridecanal, a nutrient, anantibiotic, a lysis reagent, a sterilizing reagent and/or the like. Thehousing includes a puncturer that defines a transfer pathway in fluidcommunication with the reagent volume. A delivery portion of the housingdefines a delivery pathway between the transfer pathway and the reactionchamber when the housing is coupled to the reaction chamber. In someembodiments, at least a portion of the delivery pathway can at leastpartially surround the puncturer. The actuator has a plunger portiondisposed within the reagent volume. An engagement portion of theactuator can be manipulated to move the plunger portion within thereagent volume to deform the reagent container. The puncturer can piercethe frangible portion of the reagent container to convey a reagent fromthe reagent container into the reaction chamber via the transfer pathwayand/or the delivery pathway.

In some embodiments, an apparatus includes a housing, a reagentcontainer, an actuator, and a lock member. The housing can be removablycoupled to a reaction chamber (e.g., which can contain a target cell).The housing defines a reagent volume and includes a delivery portionthat defines a delivery pathway. The delivery pathway places the reagentvolume in fluid communication with the reaction chamber when the housingis coupled to the reaction chamber. The delivery portion has a puncturerin fluid communication with the delivery pathway. The reagent containeris disposed within the reagent volume of the housing and can contain anysuitable reagent or substance. For example, the reagent container cancontain one or more transduction particles, a reagent formulated toreact with one or more reporter molecules in a sample to enhanceproduction of a signal or otherwise augment the signal or other assaycomponents, tridecanal, a nutrient, an antibiotic, a lysis reagent, asterilizing reagent and/or the like. The reagent container has afrangible portion and a skirt surrounding the frangible portion. Theactuator includes a plunger portion disposed within the reagent volume.The actuator can be manipulated to move the plunger portion within thereagent volume to deform the reagent container from a firstconfiguration to a second configuration. The puncturer is configured topierce the frangible portion of the reagent container to convey areagent from the reagent container into the reaction chamber via thedelivery pathway when the reagent container is in the secondconfiguration. The lock member can maintain the skirt in contact with ashoulder of the delivery portion of the housing when the reagentcontainer is in the second configuration to maintain a substantiallyfluid-tight seal between the skirt and the shoulder.

In some embodiments, an apparatus includes a housing, a reagentcontainer, and an actuator. The housing can be removably coupled to areaction chamber (e.g., which can contain a target cell). The housingincludes a delivery portion that defines a delivery pathway and includesa puncturer in fluid communication with the delivery pathway. Thedelivery pathway places the reagent volume in fluid communication withthe reaction chamber when the housing is coupled to the reactionchamber. The reagent container is disposed within the reagent volume ofthe housing and can contain any suitable reagent or substance. Forexample, the reagent container can contain one or more transductionparticles, a reagent formulated to react with one or more reportermolecules in a sample to enhance production of a signal or otherwiseaugment the signal or other assay components, tridecanal, a nutrient, anantibiotic, a lysis reagent, a sterilizing reagent and/or the like. Thereagent container includes a contact portion, a frangible portion, and askirt surrounding the frangible portion. The actuator has a plungerportion disposed within the reagent volume. The plunger portion cancorrespond to one or more of the contact portion of the reagentcontainer or the puncturer. The actuator can be manipulated to move theplunger portion within the reagent volume such that the plunger portionengages the contact portion of the reagent container to deform thereagent container from a first configuration to a second configuration.The puncturer is configured to pierce the frangible portion of thereagent container to convey a reagent from the reagent container intothe reaction chamber via the delivery pathway when the reagent containeris in the second configuration.

In some embodiments, a method includes disposing a sample into areaction chamber. The reaction chamber is packaged to contain a reagent(e.g., a liquid or dry composition, such as a tablet) formulated to mixwith the sample to form an assay media. One or more transductionparticles associated with a cell phenotype are mixed with the sample inthe reaction chamber. The one or more transduction particles areengineered to include a nucleic acid molecule formulated to cause thecell phenotype to produce one or more reporter molecules. The reagent isformulated to suppress production of the one or more reporter moleculesin a portion of the cell phenotype. The reagent can include any suitablesubstance. For example, the reagent can include an antibiotic and/or acolorant. A first signal associated with the reagent is received. Thefirst signal can be associated with any suitable characteristic of thereagent, such as a color of the reagent, and can therefore be used toindicate and/or confirm the presence of the reagent. The sample and theone or more transduction particles are maintained when the first signalindicates the presence of the reagent to express the one or morereporter molecules when the cell phenotype is present in the sample. Asecond signal associated with a quantity of the one or more reportermolecules is received. In some embodiments, a substance formulated toreact with the one or more reporter molecules can be disposed into thesample to generate or enhance the second signal.

In some embodiments, a method includes receiving a container thatcontains a swab and a transport media. The swab includes a shaft and acollection portion constructed from non-wound material. The transportmedia includes a sample released from the collection portion. Thecollection portion can be constructed from and/or includes any suitablematerial of non-wound construction. For example, the collection portioncan be constructed from and/or include a foam material. The transportmedia and the sample are transferred into a reaction chamber. Thetransport media is mixed in the reaction chamber with one or moretransduction particles associated with a target cell. The one or moretransduction particles are engineered to include a nucleic acid moleculeformulated to cause the target cell to produce one or more reportermolecules. The one or more reporter molecules can include any suitablesubstance. For example, the one or more reporter molecules can include abacterial luciferase, a eukaryotic luciferase, a fluorescent protein, anenzyme suitable for colorimetric detection, a protein suitable forimmunodetection, a peptide suitable for immunodetection, and/or anucleic acid that functions as an aptamer or that exhibits enzymaticactivity. The mixture of the transport media and the one or moretransduction particles is maintained at a temperature of at least 20degrees Celsius for a period of about eight hours or less to express theone or more reporter molecules when the target cell is present in thesample. A signal associated with a quantity of the one or more reportermolecules can be received.

In other embodiments, the collection portion may be added to or disposedwithin the reaction chamber directly. In such embodiments, thecollection portion may remain in the reaction chamber throughout theassay or it may be removed from the reaction chamber after the sample onthe collection portion is released into the reaction chamber.

As described herein, the terms “gene,” “DNA” and “nucleotide” mean thewhole or a portion of the genetic sequence of the target bacteria or thevector.

As described herein, the term “plasmid” means the engineered gene,sequence and/or molecule contained within the vector that includesregulatory elements, nucleic acid sequences homologous to target genes,and various reporter constructs for causing the expression of reportermolecules within a viable cell and/or when an intracellular molecule ispresent within a target cell.

A “transduction particle” refers to a virus capable of delivering anon-viral nucleic acid molecule into a cell. The virus can be abacteriophage, adenovirus, etc. A “non-replicative transductionparticle” refers to a virus capable of delivering a non-viral nucleicacid molecule into a cell, but does not package its own replicated viralgenome into the transduction particle. The virus can be a bacteriophage,adenovirus, etc.

As used herein, “reporter nucleic acid molecule” refers to a nucleotidesequence comprising a DNA or RNA molecule. The reporter nucleic acidmolecule can be naturally occurring or an artificial or syntheticmolecule. In some embodiments, the reporter nucleic acid molecule isexogenous to a host cell and can be introduced into a host cell as partof an exogenous nucleic acid molecule, such as a plasmid or vector. Incertain embodiments, the reporter nucleic acid molecule can becomplementary to a target gene in a cell. In other embodiments, thereporter nucleic acid molecule comprises a reporter gene encoding areporter molecule (e.g., reporter enzyme, protein). In some embodiments,the reporter nucleic acid molecule is referred to as a “reporterconstruct” or “nucleic acid reporter construct.”

As used herein, a “reporter molecule” or “reporter” refers to a molecule(e.g., nucleic acid or protein) that confers onto an organism adetectable or selectable phenotype. The detectable phenotype can becolorimetric, fluorescent or luminescent, for example. Reportermolecules can be expressed from reporter genes encoding enzymesmediating luminescence reactions (luxA, luxB, luxAB, luc, rue, nluc),genes encoding enzymes mediating colorimetric reactions (lacZ, HRP),genes encoding fluorescent proteins (GFP, eGFP, YFP, RFP, CFP, BFP,mCherry, near-infrared fluorescent proteins), nucleic acid moleculesencoding affinity peptides (His-tag, 3×-FLAG), and genes encodingselectable markers (ampC, tet(M), CAT, erm). The reporter molecule canbe used as a marker for successful uptake of a nucleic acid molecule orexogenous sequence (plasmid) into a cell. The reporter molecule can alsobe used to indicate the presence of a target gene, target nucleic acidmolecule, target intracellular molecule, or a cell, as described herein.Alternatively, the reporter molecule can be the reporter nucleic acidmolecule itself, such as an aptamer or ribozyme.

In some embodiments, the reporter nucleic acid molecule is operativelylinked to a promoter. In other aspects, the promoter can be chosen ordesigned to contribute to the reactivity and cross-reactivity of thereporter system based on the activity of the promoter in specific cells(e.g., specific species) and not in others. In certain aspects, thereporter nucleic acid molecule comprises an origin of replication. Inother aspects, the choice of origin of replication can similarlycontribute to reactivity and cross reactivity of the reporter system,when replication of the reporter nucleic acid molecule within the targetcell contributes to or is required for reporter signal production basedon the activity of the origin of replication in specific cells (e.g.,specific species) and not in others. In some embodiments, the reporternucleic acid molecule forms a replicon capable of being packaged asconcatameric DNA into a progeny virus during virus replication.

As used herein, the singular forms “a,” “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, the term “a member” is intended to mean a single member or acombination of members, “a material” is intended to mean one or morematerials, or a combination thereof.

As used herein, a term referring to multiple components or portionsthereof is intended to refer to a first component or a first portionthereof, and/or a second component or a second portion thereof, unlessthe context clearly dictates otherwise. Thus, for example, the term“puncturers” is intended to refer to a “first puncturer” and/or a“second puncturer.”

As used herein, the terms “about” and “approximately” generally meanplus or minus 10% of the value stated. For example, about 0.5 wouldinclude 0.45 and 0.55, about 10 would include 9 to 11, about 1000 wouldinclude 900 to 1100.

The term “fluid-tight” is understood to encompass both a hermetic seal(i.e., a seal that is gas-impervious) as well as a seal that isliquid-impervious. The term “substantially” when used in connection with“fluid-tight,” “gas-impervious,” and/or “liquid-impervious” is intendedto convey that, while total fluid imperviousness is desirable, someminimal leakage due to manufacturing tolerances, or other practicalconsiderations (such as, for example, the pressure applied to the sealand/or within the fluid), can occur even in a “substantiallyfluid-tight” seal. Thus, a “substantially fluid-tight” seal includes aseal that prevents the passage of a fluid (including gases, liquidsand/or slurries) therethrough when the seal is maintained at a constantposition and at fluid pressures of less than about 5 psig, less thanabout 10 psig, less than about 20 psig, less than about 30 psig, lessthan about 50 psig, less than about 75 psig, less than about 100 psigand all values in between. Similarly, a “substantially liquid-tight”seal includes a seal that prevents the passage of a liquid (e.g., aliquid medicament) therethrough when the seal is maintained at aconstant position and is exposed to liquid pressures of less than about5 psig, less than about 10 psig, less than about 20 psig, less thanabout 30 psig, less than about 50 psig, less than about 75 psig, lessthan about 100 psig and all values in between.

FIGS. 1-3 show a container assembly 1700 according to an embodiment in afirst configuration (FIG. 1), a second configuration (FIG. 2), and athird configuration (FIG. 3). The container assembly 1700 can be usedwith and manipulated by any of the instruments and/or any of thecomponents described herein and in U.S. patent application Ser. No.13/802,461, entitled “Systems and Methods for Detection of Cells usingEngineered Transduction Particles,” (“the '461 application”) which isincorporated herein by reference in its entirety. In this manner, thecontainer assembly 1700 and any of the container assemblies describedherein can be used to detect and/or identify target cells (e.g.,bacteria) within a sample according to any of the methods describedherein or in the '461 application. For example, in some embodiments, thecontainer assembly 1700 can be used to dispose and/or mix a reagent intoa sample while maintaining fluidic isolation between the container andan outside region. In this manner, the method of cell identification canbe performed in a closed system and/or a homogeneous assay. Similarlystated, in some embodiments the container assembly 1700 is used inmethods of cell identification and/or detection that do not involveremoval of contents from the container assembly 1700, separation of thecontents within the container assembly 1700, washing of the contentswithin the container assembly 1700 and/or rinsing of the contents withinthe container assembly 1700.

The container assembly 1700 includes a housing 1741, an actuator 1750,and a reaction chamber 1732. The housing 1741 is removably coupled tothe reaction chamber 1732. For example, in some embodiments, the housing1741 can be threadedly coupled to the reaction chamber 1732. In otherembodiments, the housing 1741 and the reaction chamber 1732 can form aninterference fit to couple the housing 1741 to the reaction chamber1732. The housing 1741 defines a reagent volume 1742 configured toreceive a reagent container 1780. The housing 1741 includes a puncturer1792 and a delivery portion 1770. In some embodiments, the housing 1741,the delivery portion 1770 and/or the puncturer 1792 can bemonolithically constructed. In other embodiments, the housing 1741, thedelivery portion 1770 and/or the puncturer 1792 can be formed separatelyand then joined together.

The puncturer 1792 is configured to pierce (e.g., rupture) a frangibleportion 1788 of the reagent container 1780 to convey a reagent from thereagent container 1780 into the reaction chamber 1732. As shown in FIGS.1-3, the puncturer 1792 includes a structure that terminates in a singlesharp point configured to pierce the reagent container 1780. Moreover,the structure of the puncturer 1792 defines a transfer pathway 1793 influid communication with the reagent volume 1742. As shown in FIG. 4, insome embodiments, the inclusion of the transfer pathway 1793 results ina discontinuous cross-sectional shape in the puncturer 1792 (thecross-sectional view is shown below or “downstream” from the singlesharp point). Thus, as described in more detail herein, when thepuncturer 1792 pierces the reagent container 1780, the transfer pathway1793 provides a pathway through which the contents of the reagentcontainer 1780 can flow. As shown, the pathway 1793 is non-parallel tothe frangible portion 1788 of the reagent container 1780. In particular,the pathway 1793 is substantially perpendicular to the frangible portion1788 of the reagent container 1780. Said another way, the pathway 1793is aligned with and/or parallel to the direction of motion of theactuator 1750 (see arrow AA). Moreover, the arrangement of the transferpathway 1793 and/or the cross-sectional shape of the puncturer 1792 canlimit clogging or obstructions that may result from the piercing, aswell as “dead volume” after actuation, thus providing a more repeatabledelivery of the contents of the reagent container 1780.

Although shown as including a single sharp point, in other embodiments,a puncture can include a sharp edge (e.g., a linear edge) and/or seriesof protrusions configured to pierce the reagent container. In suchembodiments, for example, the structure supporting or defining each ofthe series of protrusions can define a transfer pathway (similar to thetransfer pathway 1793).

Although shown as being a substantially linear pathway that is parallelto the frangible portion 1788, in other embodiments, the transferpathway 1793 can have any suitable shape, direction and/orconfiguration, such as for example, a helical shape, a tapered shape orthe like. Although the cross-sectional shape of the transfer pathway1793 is shown in FIG. 4 as being curved and/or semi-circular, in otherembodiments, the cross-sectional shape of the transfer pathway 1793 canhave any suitable shape. Moreover, the shape and/or size of the transferpathway 1793 can be variable (e.g., as a function of the distance fromthe puncturing tip). Although the puncturer 1792 is shown as including asingle transfer pathway 1793, in other embodiments, a puncturer candefine any suitable number of transfer pathways.

The delivery portion 1770 is configured to facilitate the delivery ofthe contents from the reagent container 1780 and/or the reagent volume1742 into the reaction chamber 1732. Thus, as shown, the deliveryportion 1770 can provide any suitable pathway and/or mechanism fordelivering transduction particles and/or reagents disposed in thereagent container 1780 and/or reagent volume 1742 into the reactionchamber 1732. In particular, the delivery portion 1770 defines adelivery pathway 1771 between the transfer pathway 1793 and the reactionchamber 1732. The delivery pathway 1771 can have any suitable sizeand/or shape, and can accommodate any desired flow rate therethrough.For example, in some embodiments, the transfer pathway 1793 and/or thedelivery pathway 1771 can accommodate any suitable flow rate, e.g., 1ml/sec, 2 ml/sec, 3 ml/sec, 4 ml/sec, 5 ml/sec.

The actuator 1750 has a plunger portion 1754 disposed within the reagentvolume 1742 and an engagement portion 1752. The engagement portion 1752of the actuator 1750 is configured to be manipulated to move the plungerportion 1754 within the reagent volume 1742 to deform the reagentcontainer 1780. In this manner, movement of the plunger portion 1754 canurge the frangible portion 1788 of the reagent container 1780 againstthe puncturer 1792 to pierce and/or rupture the frangible portion 1788.The plunger portion 1754 of the actuator 1750 and a portion of thehousing 1741 can collectively define a seal to fluidically and/oroptically isolate the reagent volume 1742 from a volume outside of thehousing 1741.

The reagent container 1780 can be completely or partially filled withany suitable reagent or substance. For example, the reagent container1780 can contain transduction particles that include an engineerednucleic acid formulated to cause the target cell (e.g., bacteria) toproduce one or more reporter molecules. In some embodiments, the reagentcontainer 1780 can contain one or more transduction particles engineeredto be incapable of replication (e.g., lytic replication, lysogenicreplication). For example, in some embodiments, the reagent container1780 can contain any of the transduction particles described herein andin U.S. Provisional Application Nos. 61/983,765, entitled “ReagentCartridge for Detection of Cells,” filed Apr. 24, 2014; 61/779,177,entitled “Non-Replicative Transduction Particles and TransductionParticle-Based Reporter Systems,” filed Mar. 13, 2013; 61/939,126,entitled “Systems and Methods for Packaging Nucleic Acid Molecules intoNon-Replicative Transduction Particles and Their Use as CellularReporters,” filed Feb. 12, 2014; and 61/897,040, entitled “TranscriptDetection Systems and Methods,” filed Oct. 29, 2013, and InternationalPatent Application No. PCT/US2014/026536, entitled “Non-ReplicativeTransduction Particles and Transduction Particle-Based ReporterSystems,” filed Mar. 13, 2014, each of which is incorporated herein byreference in its entirety.

In some embodiments, the reagent container can contain a reagentformulated to react with one or more reporter molecules to generateand/or enhance production of a signal. For another example, the reagentcontainer 1780 can include a substrate, such as tridecanal, that caninteract with a reporter molecule (e.g., luciferase), to produce ameasurable signal, e.g., via a luminescence reaction. For yet anotherexample, in some embodiments, the reagent container 1780 can include anutrient, an antibiotic (e.g., Beta-lactams, extended-spectrumbeta-lactams, Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, anygeneration of Cephalosporins, Glycopeptides, Lincosamides, Lipopeptide,Macrolides, Monobactams, Nitrofurans, Oxazolidonones, Penicillins,Polypeptides, Quinolones, Fluoroquinolones, Sulfonamides, Tetracyclines,mycobacterial antibiotics, Chloramphenicol, Mupirocin), a lysis reagent,a sterilizing reagent, a colorant and/or the like.

The reagent container 1780 can be shaped and sized to be disposedsubstantially inside the reagent volume 1742. The reagent container 1780can be constructed from materials that are substantially impermeable toand/or substantially chemically inert from the substance containedtherein (e.g., transduction particle, substrate, antibiotics, buffers,surfactants, or any other reagent that can be used with the detectionassay) and the outside environment. At least a portion of the reagentcontainer 1780 (e.g., the frangible portion 1788) can be constructedfrom a material (e.g., polymer film, such as any form of polypropylene)having certain temperature characteristics such that the desiredproperties and integrity are maintained over a certain temperature. Forexample, in some instances, it can be desirable to store the reagentcontainer 1780 containing reagent and/or substrate in a refrigeratedcondition. In some embodiments, a portion of the reagent container 1780can be constructed from bi-axially oriented polypropylene (BOP). In someembodiments, a portion of the reagent container 1780 can be constructedfrom aluminum. In some embodiments, a portion of the reagent container1780 can be constructed from polyvinyl chloride (PVC), ethylene vinylalcohol (EVOH), polyethylene (PE) and/or polychlorotrifluoroethene(PCTFE or PTFCE).

The reaction chamber 1732 is configured to contain a sample and/or otherreagents, and can be formed from any suitable material, for example,glass, plastic (e.g., polypropylene), acrylic, etc. In some embodiments,the reaction chamber 1732 can be formed from a lightweight, rigid and/orinert material. At least a portion of the reaction chamber 1732 (e.g.,the distal end portion) can be at least partially transparent to allowviewing, optical access and/or detection of the internal volume of thereaction chamber 1732. In some embodiments, the distal end portion ofthe reaction chamber 1732 can be polished to promote optimaltransmission of light therethrough. Although shown as being shaped as acylinder with a rounded bottom, in other embodiments, the reactionchamber 1732 can have any other suitable shape, e.g., square,rectangular, oval, polygonal, elliptical, conical, etc. For example, insome embodiments, the reaction chamber 1732 can have a substantiallyflat bottom. In some embodiments, the reaction chamber 1732 can have adiameter of 12 mm and a height of 75 mm. In some embodiments, thecontainer assembly 1700 can be provided with one or moresolutions/reagents in liquid and/or dried form (e.g., bacterial nutrientsolution, buffers, surfactants, transduction particle, colorants and/orantibiotics), predisposed within the reaction chamber 1732. In someinstances, the reaction chamber 1732 can contain any suitable reagentand/or substance. For example, in some embodiments, the reaction chamber1732 can contain one or more transduction particles, a reagentformulated to react with one or more reporter molecules in a sample togenerate and/or enhance production of a signal, a nutrient, anantibiotic, a lysis reagent, a sterilizing reagent, a colorant and/orthe like.

As shown in FIG. 1, the container assembly 1700 is in a firstconfiguration. In the first configuration, the actuator 1750 ispositioned such that the reagent container 1780 disposed within thehousing 1741 is substantially undeformed. Similarly stated, the actuator1750 is positioned such that it does not cause puncturer 1792 to piercethe reagent container 1780. Thus, the container assembly 1700 is in a“ready” state when in the first configuration. In some embodiments, thecontainer assembly 1700 can include a safety mechanism (not shown) toprevent and/or limit movement of the actuator 1750 relative to thehousing 1741 until desired by the operator.

To actuate the container assembly 1700, a force is applied to theengagement portion 1752 of the actuator 1750, thus causing the actuator1750 to move as shown by the arrow AA in FIG. 2. As shown in FIG. 2, thecontainer assembly 1700 is in a second (or “intermediate”)configuration. In the second configuration, the actuator 1750 ispositioned such that the reagent container 1780 is partially deformed.Similarly stated, the actuator 1750 is positioned such that at least aportion of the force is transferred to the reagent container 1780. Assuch, at least a portion of the reagent container 1780 becomes deformed.In some instances, in the second configuration, the puncturer 1792 canat least partially pierce a portion (e.g., the frangible portion 1788)of the reagent container 1780, thereby placing the internal volume ofthe reagent container 1780 in fluid communication with the transferpathway 1793 and/or the delivery pathway 1771.

As shown in FIG. 3, the container assembly 1700 is in a third (or“deployed”) configuration. In the third configuration, the actuator 1750is positioned such that the reagent container 1780 is substantiallydeformed. Similarly stated, the actuator 1750 is positioned such that atleast a portion of the force is transferred to the reagent container1780. In such a configuration, the puncturer 1792 has pierced thereagent container 1780 (e.g., the frangible portion 1788), such that thecontents of the reagent container have substantially exited the reagentcontainer 1780 and entered the delivery portion 1770 and/or the reactionchamber 1732 via the transfer pathway 1793, as shown by the arrow BB.

In use, the actuator 1750 (e.g., the engagement portion 1752) ismanipulated to move the plunger portion 1754 within the housing 1741such that the plunger portion 1754 engages a contact portion (notidentified in FIGS. 1-3) of the reagent container 1780 to partiallydeform the reagent container 1780 from the first configuration to thesecond configuration. As the plunger portion 1754 engages the contactportion of the reagent container 1780, the puncturer 1792 pierces aportion of the reagent container 1780 (e.g., a frangible portion 1788)to convey a reagent from the reagent container 1780 into the reactionvolume 1742, the delivery portion 1770, and/or the reaction chamber1732, at least in part via the transfer pathway 1793. From the secondconfiguration to the third configuration, the actuator 1750 ismanipulated to move the plunger portion 1754 within the housing 1741such that the plunger portion 1754 engages a contact portion of thereagent container 1780 to deform the reagent container 1780 from thesecond configuration to the third configuration. As the reagentcontainer 1780 deforms from the second configuration to the thirdconfiguration, substantially all of its contents (e.g., a reagent) isconveyed from the reagent container 1780 into the reaction volume 1742,the delivery portion 1770, and/or the reaction chamber 1732, such that“dead volume” in the reagent container 1780 is limited. In this manner,substantially repeatable delivery of the contents from the reagentcontainer 1780 to the reaction chamber 1732 can be obtained. Forexample, in some embodiments, a deformation of a first reagent containerat a first time and a deformation of a second reagent container at asecond time after the first time can be substantially similar, therebyallowing for substantially all of the contents to be transferred fromthe reagent container 1780 at the first time and the second time.Moreover, this arrangement can limit clogging or obstructions that mayresult from the piercing of the reagent container 1780, thus providing amore repeatable delivery of the contents of the reagent container 1780.

FIG. 5 shows a container assembly 2700 according to an embodiment. Thecontainer assembly 2700 can be used with and manipulated by any of theinstruments and/or any of the components described herein and in the'461 application, which is incorporated herein by reference in itsentirety. In this manner, the container assembly 2700 and any of thecontainer assemblies described herein can be used to detect and/oridentify target cells (e.g., bacteria) within a sample according to anyof the methods described herein or in the '461 application. For example,in some embodiments, the container assembly 2700 can be used to disposeand/or mix a reagent into a sample while maintaining fluidic isolationbetween the container and an outside region. In this manner, the methodof cell identification can be performed in a closed system and/or ahomogeneous assay. Similarly stated, in some embodiments the containerassembly 2700 is used in methods of cell identification and/or detectionthat do not involve removal of contents from the container assembly2700, separation of the contents within the container assembly 2700,washing of the contents within the container assembly 2700 and/orrinsing of the contents within the container assembly 2700.

The container assembly 2700 includes a housing 2741, a reaction chamber2732, a reagent container 2780, an actuator 2750, and a lock member2772. The housing 2741 is removably coupled to the reaction chamber2732. For example, in some embodiments, the housing 2741 can bethreadedly coupled to the reaction chamber 2732. In other embodiments,the housing 2741 and the reaction chamber 2732 can form an interferencefit to couple the housing 2741 to the reaction chamber 2732. The housing2741 defines a reagent volume 2742 and includes a delivery portion 2770.The delivery portion 2770 includes a puncturer 2792. In someembodiments, the housing 2741, the delivery portion 2770, and/or thepuncturer 2792 can be monolithically constructed. In other embodiments,the housing 2741, the delivery portion 2770, and/or the puncturer 2792can be formed separately and then joined together.

The delivery portion 2770 is configured to facilitate the delivery ofthe contents from the reagent container 2780 and/or the reagent volume2742 into the reaction chamber 2732. Thus, as shown, the deliveryportion 2770 can provide any suitable pathway and/or mechanism fordelivering contents disposed in the reagent container 2780 and/orreagent volume 2742 into the reaction chamber 2732. In particular, thedelivery portion 2770 defines a delivery pathway 2771 between thereagent volume 2742 and the reaction chamber 2732. The delivery pathway2771 can have any suitable size and/or shape, and can accommodate anydesired flow rate therethrough. For example, in some embodiments, thedelivery pathway 2771 can accommodate any suitable flow rate, e.g., 1ml/sec, 2 ml/sec, 3 ml/sec, 4 ml/sec, 5 ml/sec. Moreover, the shapeand/or size of the delivery pathway 2771 can be variable. Although thedelivery portion 2770 is shown as including a single delivery pathway2771, in other embodiments, a delivery portion can define any suitablenumber of delivery pathways.

Moreover, the delivery portion 2770 (and any of the delivery portionsdescribed herein) can include any suitable features, such as thedelivery pathway 2771, surface geometry, surface coating or the like.For example, as shown, the delivery portion 2770 includes a concavesurface 2774. In this manner, the delivery portion 2770 can facilitatethe delivery of the contents from the reagent container 2780 and/or thereagent volume 2742 into the reaction chamber 2732. For example, thecontents of the reagent container 2780 can be transferred along (e.g.,based at least in part on gravitational force) the concave surface ofthe delivery portion 2770 and into the reaction chamber 2732 via thedelivery pathway 2771. In other embodiments, however, the deliveryportion 2770 need not have a concave surface.

The puncturer 2792 of the delivery portion 2770 is configured to pierce(e.g., rupture) a frangible portion 2788 of the reagent container 2780to convey a reagent from the reagent container 2780 into the reactionchamber 2732. Thus, the puncturer 2792 can include any sharp point,sharp edge and/or series of protrusions configured to pierce the reagentcontainer 2780. In some embodiments, the arrangement of and/or the shapeof the puncturer can limit clogging and/or obstructions that may resultfrom the piercing, thus providing a more repeatable delivery of thecontents of the reagent container 2780. For example, in someembodiments, the puncturer 2792 can define one or more transferpathways, similar to those shown and described herein (e.g., in FIGS.1-3). Thus, the puncturer 2792 is in fluid communication with thedelivery pathway 2771 of the delivery portion 2770. In this manner, andas described in more detail herein, the puncturer can facilitate thetransfer of the contents of the reagent container 2780 to the reactionchamber 2732.

The reagent container 2780 can be completely or partially filled withany suitable reagent or substance. For example, the reagent container2780 can contain transduction particles that include an engineerednucleic acid formulated to cause the target cell (e.g., bacteria) toproduce one or more reporter molecules. In some embodiments, the reagentcontainer 2780 can contain one or more transduction particles engineeredto be incapable of replication (e.g., lytic replication, lysogenicreplication). For example, in some embodiments, the reagent container2780 can contain any of the transduction particles described herein andin U.S. Provisional Application Nos. 61/983,765, entitled “ReagentCartridge for Detection of Cells,” filed Apr. 24, 2014; 61/779,177,entitled “Non-Replicative Transduction Particles and TransductionParticle-Based Reporter Systems,” filed Mar. 13, 2013; 61/939,126,entitled “Systems and Methods for Packaging Nucleic Acid Molecules intoNon-Replicative Transduction Particles and Their Use as CellularReporters,” filed Feb. 12, 2014; and 61/897,040, entitled “TranscriptDetection Systems and Methods,” filed Oct. 29, 2013, and InternationalPatent Application No. PCT/US2014/026536, entitled “Non-ReplicativeTransduction Particles and Transduction Particle-Based ReporterSystems,” filed Mar. 13, 2014, each of which is incorporated herein byreference in its entirety.

In some embodiments, the reagent container 2780 can contain a reagentformulated to react with one or more reporter molecules to enhanceproduction of a signal. For another example, the reagent container 2780can include a substrate, such as tridecanal, that can interact with areporter molecule (e.g., luciferase), to produce a measurable signal,e.g., via a luminescence reaction. For yet another example, in someembodiments, the reagent container 2780 can include a nutrient, anantibiotic (e.g., Beta-lactams, extended-spectrum beta-lactams,Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, any generation ofCephalosporins, Glycopeptides, Lincosamides, Lipopeptide, Macrolides,Monobactams, Nitrofurans, Oxazolidonones, Penicillins, Polypeptides,Quinolones, Fluoroquinolones, Sulfonamides, Tetracyclines, mycobacterialantibiotics, Chloramphenicol, Mupirocin), a lysis reagent, a sterilizingreagent, a colorant and/or the like.

The reagent container 2780 can be shaped and sized to be disposedsubstantially inside the reagent volume 2742. The reagent container 2780can be constructed from materials that are substantially impermeable toand/or substantially chemically inert from the substance containedtherein, e.g., transduction particle, substrate, antibiotics, buffers,surfactants, or any other reagent that can be used with the detectionassay. At least a portion of the reagent container 2780 (e.g., thefrangible portion 2788) can be constructed from a material (e.g.,polymer film, such as any form of polypropylene) having certaintemperature characteristics such that the desired properties andintegrity are maintained over a certain temperature. For example, insome instances, it can be desirable to store the reagent container 2780containing reagent and/or substrate in a refrigerated condition. In someembodiments, a portion of the reagent container 2780 can be constructedfrom bi-axially oriented polypropylene (BOP). In some embodiments, aportion of the reagent container 2780 can be constructed from aluminum.In some embodiments, a portion of the reagent container 2780 can beconstructed from polyvinyl chloride (PVC), ethylene vinyl alcohol(EVOH), polyethylene (PE) and/or polychlorotrifluoroethene (PCTFE orPTFCE).

The reagent container 2780 has a skirt 2781 and a frangible portion2788. The skirt 2781 surrounds at least a portion of the frangibleportion 2788. As shown, the skirt 2781 is disposed between the lockmember 2772 and a shoulder portion 2775 of the delivery portion 2770 ofthe housing 2741. In this manner, and as discussed in further detailherein, the skirt 2781 can be secured (e.g., grabbed, grasped, held,pinched, interference fitted, etc.) at least in part by the lock member2772. As such, the skirt 2781 can provide a securement function suchthat a position of the reagent container 2780 can be substantiallymaintained during use. The skirt 2781 can be any suitable size and/orshape, and can include any suitable surface design (e.g., smooth, roughand/or the like). For example, in some embodiments, the skirt 2781 canbe sized and/or shaped to correspond to a portion of the lock member2772.

The actuator 2750 has a plunger portion 2754 disposed within the reagentvolume 2742 and an engagement portion 2752. The engagement portion 2752of the actuator 2750 is configured to be manipulated to move the plungerportion 2754 within the reagent volume 2742 to deform the reagentcontainer 2780 from a first configuration to a second configuration (thesecond configuration is not shown in FIG. 5). In this manner, movementof the plunger portion 2754 can urge the frangible portion 2788 of thereagent container 2780 against the puncturer 2792 to pierce and/orrupture the frangible portion 2788. Thus, as described in more detailherein, when the puncturer 2792 pierces the reagent container 2780, thedelivery pathway 2771 provides a pathway through which the contents ofthe reagent container 2780 can flow (e.g., when in the secondconfiguration). The plunger portion 2754 of the actuator 2750 and aportion of the housing 2741 can collectively define a seal tofluidically and/or optically isolate the reagent volume 2742 from avolume outside of the housing 2741.

As described above, the lock member 2772 is configured to maintain atleast a portion of the skirt 2781 of the reagent container 2780 incontact with the delivery portion 2770 (e.g., the shoulder 2775 of thedelivery portion 2770) of the housing 2741. Moreover, the portion of theskirt 2781 and the shoulder 2775 can form a substantially fluid-tightseal, thus reducing and/or eliminating backflow of the reagent withinthe reagent container 2780 during use. In this manner, by maintainingthe position of the skirt 2781 relative to the delivery portion 2770,the lock member 2772 can facilitate maintaining the substantiallyfluid-tight seal between the skirt 2781 and the shoulder 2775 of thedelivery portion 2770. In addition, the lock member 2772 can limitmovement of the reagent container 2780 relative to the delivery portion.In particular, the lock member 2772 can limit movement when the reagentcontainer 2780 is deformed from a first configuration to a secondconfiguration. In this manner, in use, the lock member 2772 can limitand/or prevent undesired movement of the reagent container 2780, therebyproviding for substantial repeatable delivery of the contents from thereagent container 2780 to the reaction chamber 2732. Similarly stated,the reagent container 2780 can be held in a preferable position (e.g.,stabilized) when in the first configuration and/or the secondconfiguration.

The lock member 2772 can be any suitable size and/or shape. For example,in some embodiments, the lock member 2772 can be sized and/or shaped tocorrespond to (e.g., by shape, size, surface design, texture, etc.) aportion of the skirt 2781 of the reagent container 2780. In this manner,the lock member 2772 and the skirt 2781 can cooperatively function tosubstantially maintain the reagent container 2780 in a desired positionrelative to the delivery portion 2770.

FIGS. 6 and 7 show a container assembly 3700 according to an embodimentin a first configuration (FIG. 6) and a second configuration (FIG. 7).The container assembly 3700 can be used with and manipulated by any ofthe instruments and/or any of the components described herein and in the'461 application, which is incorporated herein by reference in itsentirety. In this manner, the container assembly 3700 and any of thecontainer assemblies described herein can be used to detect and/oridentify target cells (e.g., bacteria) within a sample according to anyof the methods described herein or in the '461 application. For example,in some embodiments, the container assembly 3700 can be used to disposeand/or mix a reagent into a sample while maintaining fluidic isolationbetween the container and an outside region. In this manner, the methodof cell identification can be performed in a closed system and/or ahomogeneous assay. Similarly stated, in some embodiments the containerassembly 3700 is used in methods of cell identification and/or detectionthat do not involve removal of contents from the container assembly3700, separation of the contents within the container assembly 3700,washing of the contents within the container assembly 3700 and/orrinsing of the contents within the container assembly 3700.

The container assembly 3700 includes a housing 3741, a reaction chamber3732, a reagent container 3780, and an actuator 3750. The housing 3741is removably coupled to the reaction chamber 3732. For example, in someembodiments, the housing 3741 can be threadedly coupled to the reactionchamber 3732. In other embodiments, the housing 3741 and the reactionchamber 3732 can form an interference fit to couple the housing 3741 tothe reaction chamber 3732. The housing 3741 defines a reagent volume3742 and includes a delivery portion 3770. The delivery portion 3770includes a puncturer 3792. In some embodiments, the housing 3741, thedelivery portion 3770, and/or the puncturer 3792 can be monolithicallyconstructed. In other embodiments, the housing 3741, the deliveryportion 3770, and/or the puncturer 3792 can be formed separately andthen joined together.

The delivery portion 3770 is configured to facilitate the delivery ofthe contents from the reagent container 3780 and/or the reagent volume3742 into the reaction chamber 3732. Thus, as shown, the deliveryportion 3770 can provide any suitable pathway and/or mechanism fordelivering contents disposed in the reagent container 3780 and/orreagent volume 3742 into the reaction chamber 3732. For example, thecontents of the reagent container 3780 can be transferred along (e.g.,urged at least in part by gravitational force, force applied by theactuator 3750, surface tension forces or the like) one or more surfacesof the delivery portion 3770 and into the reaction chamber 3732. Inparticular, the delivery portion 3770 defines a delivery pathway 3771between the reagent volume 3742 and the reaction chamber 3732. Thedelivery pathway 3771 can have any suitable size and/or shape, and canaccommodate any desired flow rate therethrough. For example, in someembodiments, the delivery pathway 3771 can accommodate any suitable flowrate, e.g., 1 ml/sec, 2 ml/sec, 3 ml/sec, 4 ml/sec, 5 ml/sec. Moreover,the shape and/or size of the delivery pathway 3771 can be variable.Although the delivery portion 3770 is shown as including a singledelivery pathway 3771, in other embodiments, a delivery portion candefine any suitable number of delivery pathways.

The puncturer 3792 of the delivery portion 3770 is configured to pierce(e.g., rupture) a frangible portion 3788 of the reagent container 3780to convey a reagent from the reagent container 3780 into the reactionchamber 3732. Thus, the puncturer 3792 can include any sharp point,sharp edge and/or series of protrusions configured to pierce the reagentcontainer 3780. The arrangement of and/or the shape of the puncturer canlimit clogging and/or obstructions that may result from the piercing,thus providing a more repeatable delivery of the contents of the reagentcontainer 3780. The puncturer 3792 can place the reagent container 3780in fluid communication with the delivery pathway 3771 of the deliveryportion 3770. In this manner, and as described in more detail herein,the puncturer can facilitate the transfer of the contents of the reagentcontainer 3780 to the reaction chamber 3732.

The reagent container 3780 can be completely or partially filled withany suitable reagent or substance. For example, the reagent container3780 can contain transduction particles that include an engineerednucleic acid formulated to cause the target cell (e.g., bacteria) toproduce one or more reporter molecules. In some embodiments, the reagentcontainer 3780 can contain one or more transduction particles engineeredto be incapable of replication (e.g., lytic replication, lysogenicreplication). For example, in some embodiments, the reagent container3780 can contain any of the transduction particles described herein andin U.S. Provisional Application Nos. 61/983,765, entitled “ReagentCartridge for Detection of Cells,” filed Apr. 24, 2014; 61/779,177,entitled “Non-Replicative Transduction Particles and TransductionParticle-Based Reporter Systems,” filed Mar. 13, 2013; 61/939,126,entitled “Systems and Methods for Packaging Nucleic Acid Molecules intoNon-Replicative Transduction Particles and Their Use as CellularReporters,” filed Feb. 12, 2014; and 61/897,040, entitled “TranscriptDetection Systems and Methods,” filed Oct. 29, 2013, and InternationalPatent Application No. PCT/US2014/026536, entitled “Non-ReplicativeTransduction Particles and Transduction Particle-Based ReporterSystems,” filed Mar. 13, 2014, each of which is incorporated herein byreference in its entirety.

In some embodiments, the reagent container can contain a reagentformulated to react with one or more reporter molecules to enhanceproduction of a signal. For another example, the reagent container 3780can include a substrate, such as tridecanal, that can interact with areporter molecule (e.g., luciferase), to produce a measurable signal,e.g., via a luminescence reaction. For yet another example, in someembodiments, the reagent container 3780 can include a nutrient, anantibiotic (e.g., Beta-lactams, extended-spectrum beta-lactams,Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, any generation ofCephalosporins, Glycopeptides, Lincosamides, Lipopeptide, Macrolides,Monobactams, Nitrofurans, Oxazolidonones, Penicillins, Polypeptides,Quinolones, Fluoroquinolones, Sulfonamides, Tetracyclines, mycobacterialantibiotics, Chloramphenicol, Mupirocin), a lysis reagent, a sterilizingreagent, a colorant and/or the like.

The reagent container 3780 can be shaped and sized to be disposedsubstantially inside the reagent volume 3742. The reagent container 3780can be constructed from materials that are substantially impermeable toand/or substantially chemically inert from the substance containedtherein, e.g., transduction particle, substrate, antibiotics, buffers,surfactants, or any other reagent that can be used with the detectionassay. At least a portion of the reagent container 3780 (e.g., thefrangible portion 3788) can be constructed from a material (e.g.,polymer film, such as any form of polypropylene) having certaintemperature characteristics such that the desired properties andintegrity are maintained over a certain temperature. For example, insome instances, it can be desirable to store the reagent container 3780containing reagent and/or substrate in a refrigerated condition. In someembodiments, a portion of the reagent container 3780 can be constructedfrom bi-axially oriented polypropylene (BOP). In some embodiments, aportion of the reagent container 3780 can be constructed from aluminum.In some embodiments, a portion of the reagent container 3780 can beconstructed from polyvinyl chloride (PVC), ethylene vinyl alcohol(EVOH), polyethylene (PE) and/or polychlorotrifluoroethene (PCTFE orPTFCE).

The reagent container 3780 has a skirt 3781, a contact portion 3782 anda frangible portion 3788. The skirt 3781 surrounds at least a portion ofthe frangible portion 3788. The skirt 3781 can be any suitable sizeand/or shape, and can include any suitable surface design (e.g., smooth,rough and/or the like). In some embodiments, the skirt 3781 and thedelivery portion 3771 can form a substantially fluid-tight seal tominimize dead volume during use.

As described below, the contact portion 3782 of the reagent container3780 is configured to contact the plunger portion 3754 of the actuator3750. The contact portion can be any suitable size and/or shape. Forexample, in some embodiments, the contact portion 3782 can be sizedand/or shaped to correspond to the actuator 3750. In other embodiments,the contact portion 3782 can include one or more stress concentrationrisers, perforations or the like to facilitate deformation of thecontact portion 3782 and/or the reagent container 3780 in a desiredmanner. For example, in some embodiments, the contact portion 3782 caninclude geometric features and/or material properties to facilitatedeformation of the reagent container 3780 in a particular directionand/or manner to minimize dead volume during use.

For example, as shown in FIG. 9, in some embodiments, the contactportion 3782, the skirt 3781 and/or the frangible portion 3788 can becollectively configured such that when a force is applied to the contactportion 3782 (as shown by the arrow EE), the frangible portion 3788 willbulge outwardly to produce a convex shape. In some embodiments, thedelivery portion 3770 of the housing 3741 can include a concave surface(not shown in FIGS. 6-9) that corresponds to the deformed or “bulged”shape of the frangible portion during use. In this manner, the deformedportion of the reagent container 3780 can be matingly received withinthe delivery portion 3770 of the housing to minimize dead volume,facilitate repeatable delivery of reagents or the like.

As shown in FIG. 8, in some embodiments, the contact portion 3782 can bean annular ring. In this manner, the contact portion 3782 can beconfigured to mate with the plunger portion 3754 of the actuator 3750substantially uniformly across an area defined by the annular ring,resulting in minimization of dead volume and repeatable conveyance ofthe contents of the reagent container 3780 to the reaction chamber 3732.Although shown to have a constant width, in other embodiments, theannular ring can be any suitable size or shape (e.g., circular, oval,rectangular etc.). For example, in some embodiments, the annular ringcan have varying dimensions (e.g., varying ring width). In this manner,the contact portion 3782 and the plunger portion 3754 can cooperativelyfunction to suitably mate, resulting in repeatable delivery of thecontents of the reagent container 3780 from the reagent container 3780.

The actuator 3750 has a plunger portion 3754 disposed within the reagentvolume 3742, and an engagement portion 3752. The engagement portion 3752of the actuator 3750 is configured to be manipulated to move the plungerportion 3754 within the reagent volume 3742 to deform the reagentcontainer 3780 from a first configuration (FIG. 6) to a secondconfiguration (FIG. 7). Similarly stated, the plunger portion 3754, inresponse to the manipulation of the engagement portion 3752 of theactuator 3750, can engage the contact portion 3782 of the reagentcontainer 3780 to deform the reagent container 3780 from the firstconfiguration to the second configuration. In this manner, movement ofthe plunger portion 3754 can urge the frangible portion 3788 of thereagent container 3780 against the puncturer 3792 to pierce and/orrupture the frangible portion 3788. Thus, as described in more detailherein, when the puncturer 3792 pierces the reagent container 3780, thedelivery pathway 3771 provides a pathway through which the contents ofthe reagent container 3780 can flow (e.g., when in the secondconfiguration).

In some embodiments, the plunger portion 3754 of the actuator 3750 and aportion of the housing 3741 can collectively define a seal tofluidically and/or optically isolate the reagent volume 3742 from avolume outside of the housing 3741. Moreover, the plunger portion 3754can any suitable size and/or shape. For example, the plunger portion3754 can be shaped and/or sized to correspond to the contact portion3782 of the reagent container 3780 and/or to the puncturer 3792. Furtherto this example, as shown, the plunger portion 3754 has a curved shapeconfigured to matingly engage with a curved shape of the contact portion3782 of the reagent container 3780. In this manner, the plunger portion3754, the contact portion 3782, and/or the puncturer 3792 can matetogether and/or cooperatively function to limit dead volume (e.g., deadvolume within the delivery portion 3770). Minimizing dead volume allowsfor repeatable conveyance of the contents of the reagent container 3780to the reaction chamber 3732, and repeatable piercing of the reagentcontainer 3780 (e.g., repeatable blister burst).

As shown in FIG. 6, the container assembly 3700 is in a firstconfiguration. In the first configuration, the actuator 3750 ispositioned such that the reagent container 3780 disposed within thehousing 3741 is substantially undeformed. Similarly stated, the actuator3750 is positioned such that it does not cause puncturer 3752 to piercethe reagent container 3780. Thus, the container assembly 3700 is in a“ready” state when in the first configuration. In some embodiments, thecontainer assembly 3700 can include a safety mechanism (not shown) toprevent and/or limit movement of the actuator 3750 relative to thehousing 3741 until desired by the operator.

To actuate the container assembly 3700, a force is applied to theengagement portion 3752 of the actuator 3750, thus causing the actuator3750 to move as shown by the arrow CC in FIG. 6. As shown in FIG. 7, thecontainer assembly 3700 is in a second configuration. In the secondconfiguration, the actuator 3750 is positioned such that the reagentcontainer 3780 is substantially deformed. Similarly stated, the actuator3750 is positioned such that at least a portion of the force istransferred to the reagent container 3780. In such a configuration, thepuncturer 3792 has pierced the reagent container 3780 (e.g., thefrangible portion 3788), such that the contents of the reagent containerhave substantially exited the reagent container 3780 and entered thedelivery portion 3770 and/or the reaction chamber 3732, as shown by thearrow DD.

In use, the actuator 3750 (e.g., the engagement portion 3752) ismanipulated to move the plunger portion 3754 within the housing 3741such that the plunger portion 3754 engages a contact portion of thereagent container 3780 to partially deform the reagent container 3780from the first configuration to the second configuration. As the plungerportion 3754 engages the reagent container 3780, the puncturer 3792pierces a portion of the reagent container 3780 (e.g., a frangibleportion 3788) to convey a reagent from the reagent container 3780 intothe reaction volume 3742, the delivery portion 3770, and/or the reactionchamber 3732. From the first configuration to the second configuration,the actuator 3750 is manipulated to move the plunger portion 3754 withinthe housing 3741 such that the plunger portion 3754 engages a contactportion of the reagent container 3780 to deform the reagent container3780. As the reagent container 3780 deforms, substantially all of itscontents (e.g., a reagent) is conveyed from the reagent container 3780into the reaction volume 3742, the delivery portion 3770, and/or thereaction chamber 3732, such that “dead volume” in the reagent container3780 is limited. In this manner, substantially repeatable delivery ofthe contents from the reagent container 3780 to the reaction chamber3732 can be obtained. For example, in some embodiments, a deformation ofa first reagent container at a first time and a deformation of a secondreagent container at a second time after the first time can besubstantially similar, thereby allowing for substantially all of thecontents to be transferred from the reagent container 3780 at the firsttime and the second time. Moreover, this arrangement can limit cloggingor obstructions that may result from the piercing of the reagentcontainer 3780, thus providing a more repeatable delivery of thecontents of the reagent container 3780.

Although the container assemblies 1700, 2700 and 3700 are shown asincluding only one reagent container, in other embodiments, a housingand/or container assembly can include any suitable number of reagentcontainers. For example, FIGS. 10 and 11 show a perspective view of acontainer assembly 4700 and an exploded view of the container assembly4700, respectively, according to an embodiment. The container assembly4700 can be used with and manipulated by any of the instruments and/orany of the components described herein and in U.S. patent applicationSer. No. 13/802,461, entitled “Systems and Methods for Detection ofCells using Engineered Transduction Particles,” which is incorporatedherein by reference in its entirety. In this manner, the containerassembly 4700 and any of the container assemblies described herein canbe used to detect and/or identify target cells (e.g., bacteria) within asample according to any of the methods described herein or in the '461application. For example, in some embodiments, the container assembly4700 can be used to dispose and/or mix a reagent into a sample whilemaintaining fluidic isolation between the container and an outsideregion. In this manner, the method of cell identification can beperformed in a closed system and/or a homogeneous assay. Similarlystated, in some embodiments the container assembly 4700 is used inmethods of cell identification and/or detection that do not involveremoval of contents from the container assembly 4700, separation of thecontents within the container assembly 4700, washing of the contentswithin the container assembly 4700 and/or rinsing of the contents withinthe container assembly 4700.

The container assembly 4700 includes a housing 4741, a first actuator4750, a second actuator 4760, and a reaction chamber 4732. The assemblyof the housing 4741, the first actuator 4750, the first reagentcontainer 4780, the second actuator 4760 and the second reagentcontainer 4790 can be referred to as a “cap assembly” or “reagentassembly.” The housing 4741 (and/or the cap assembly) is removablycoupled to the reaction chamber 4732. For example, as shown in FIG. 10,the housing 4741 can be threadedly coupled to a proximal portion of thereaction chamber 4732. In other embodiments, the housing 4741 and thereaction chamber 4732 can form an interference fit to couple the housing4741 to the reaction chamber 4732. Thus, the housing 4741 (or capassembly) can be stored separately from and/or spaced apart from thereaction chamber 4732. In this manner, a user can then dispose a sampleinto the reaction chamber 4732 in accordance with the methods describedherein (and in the '461 application, which is incorporated herein byreference in its entirety), and can then assembly the housing 4741 (orcap assembly) to the reaction chamber 4732 (or “tube”) and complete thesteps for cell identification, as described herein.

The housing 4741 defines a first reagent volume 4742 configured toreceive a first reagent container 4780 and a second reagent volume 4744configured to receive a second reagent container 4790. The housing 4741includes a first puncturer 4792, a second puncturer 4794, a firstdelivery portion 4770, and a second delivery portion 4772. In someembodiments, the housing 4741, the first delivery portion 4770, thesecond delivery portion 4772, the first puncturer 4792, and/or thesecond puncturer 4794 can be monolithically constructed. In otherembodiments, the housing 4741, the first delivery portion 4770, thesecond delivery portion 4772, the first puncturer 4792, and/or thesecond puncturer 4794 can be formed separately and then joined together.

FIGS. 12-14 show a view of an interior portion of the housing 4741, across-sectional side view taken along line X-X in FIG. 12, and adetailed view of the cross-sectional side view shown in FIG. 13,respectively. As shown, the housing 4741 defines a first reagent volume4742 configured to receive the first reagent container 4780 (not shown)and a second reagent volume 4744 configured to receive the secondreagent container 4790 (not shown). In addition, as shown, the firstdelivery portion 4770 defines a first delivery pathway 4771 in fluidcommunication with the first puncturer 4792. Similarly, the seconddelivery portion 4772 defines a second delivery pathway 4773 in fluidcommunication with the second puncturer 4794.

The first puncturer 4792 and/or the second puncturer 4794 are configuredto pierce (e.g., rupture) the first frangible portion 4788 of thereagent container 4780 (not shown in FIG. 12) and the second frangibleportion of the reagent container 4790 (not shown in FIG. 12),respectively, to convey reagent from the reagent container 4780 and/orthe reagent container 4790 into the reaction chamber 4732. Thus, thepuncturer 4792 and the puncturer 4794 include a sharp point, sharp edgeand/or a protrusion, as shown, to pierce the reagent container 4780 andthe reagent container 4790, respectively. Moreover, the first puncturer4792 defines a first series of transfer pathways 4793 in fluidcommunication with the first reagent volume 4742, and the secondpuncturer 4794 defines a second series of transfer pathways 4795 influid communication with the second reagent volume 4744. In particular,each of the first series of transfer pathways 4793 and the second seriesof transfer pathways 4795 includes four channels spaced at approximately90 degree intervals about the center point of the respective puncturer.Thus, as shown, the inclusion of the first series of transfer pathways4793 and/or the second series of transfer pathways 4795 produces adiscontinuous cross-sectional shape in the first puncturer 4792 and thesecond puncturer, respectively 4794. When the first puncturer 4792pierces the first reagent container 4780, the first series of transferpathways 4793 provides pathways through which the contents of thereagent container 4780 can flow. Similarly, when the second puncturer4794 pierces the second reagent container 4790, the second series oftransfer pathways 4795 provides pathways through which the contents ofthe reagent container 4790 can flow. Moreover, the arrangement of thefirst series of transfer pathways 4793, the second series of transferpathways 4795, the cross-sectional shape of the first puncturer 4792,and/or the cross-sectional shape of the second puncturer 4794 can limitclogging or obstructions that may result from the piercing, thusproviding a more repeatable delivery of the contents of the firstreagent container 4780 and/or the second reagent container 4790.

As shown, the puncturer 4792 and/or the puncturer 4794 are disposedalong and/or aligned with an axial centerline of the reagent volume 4742and the reagent volume 4744, respectively. Similarly stated, thepuncturer 4792 and the puncturer 4794 are centered with respect to thereagent container 4780 and the reagent container 4790, respectively.Such a configuration promotes repeatable, substantially completedelivery of the contents from the reagent container 4780 and/or thereagent container 4790, as described herein. In other embodiments,however, the puncturer 4792 and/or the puncturer 4794 can be offset froman axial centerline of the reagent volume 4742 and the reagent volume4744, respectively. In such embodiments, for example, the offset can bebased on a shape, size, slope, and/or configuration of the firstdelivery portion 4770, the second delivery portion 4772, and/or thereaction chamber 4732. For example, in some embodiments, the puncturer4792 can be offset laterally towards a side portion of the housing 4741.Similarly, in some embodiments, the puncturer 4794 can be offsetlaterally towards a side portion of the housing 4741. In this manner,the contents of the reagent container 4780 and/or the reagent container4790 can be encouraged to flow relatively close to the sidewall portion4734, thus preventing splash and/or turbulence of the contents. Thus, insuch embodiments, an offset of the puncturer 4792 and/or the puncturer4794 can provide efficient, desirable and/or complete delivery ofcontents from the reagent container 4780 and the reagent container 4790,respectively.

Although the cross-sectional shapes of the first series of transferpathways 4793 and the second series of transfer pathways 4795 are shownin FIG. 12 as being curved and/or semi-circular, in other embodiments,the first series of transfer pathways 4793 and/or the second series oftransfer pathways 4795 can have any suitable shape and configuration,such as for example, a helical shape, a tapered shape and/or the like.Moreover, although the shape and/or size of the first series of transferpathways 4793 and/or the second series of transfer pathways 4795 areshown in FIG. 13 as having a vertical orientation and a constantdiameter (cross-sectional area, flow area), in other embodiments thefirst series of transfer pathways 4793 and/or the second series oftransfer pathways 4795 can have any suitable orientation, configuration,and size. For example, in some embodiments, the first series of transferpathways 4793 and/or the second series of transfer pathways 4795 canhave varying cross-sectional (or flow) areas (e.g., as a function of thedistance from the puncturing tip) and/or non-vertical orientations(e.g., sloped). In this manner, the first series of transfer pathways4793 and/or the second series of transfer pathways 4795 can beconfigured to promote a controlled and/or desired flow rate of thesubstances flowing therethrough. Moreover, although the first series oftransfer pathways 4793 and the second series of transfer pathways 4795are each shown in FIG. 12 as defining four channels, in otherembodiments, a transfer pathway can define any suitable number oftransfer channels.

FIGS. 13 and 14 show a cross-sectional view and a close-upcross-sectional view, respectively, of the housing 4741 shown in FIG.12. As shown, the first delivery pathway 4771 is in fluid communicationwith the first series of transfer pathways 4793, the first reagentvolume 4742, and the reaction chamber 4732. Similarly, the seconddelivery pathway 4773 is in fluid communication with the second seriesof transfer pathways 4795, the second reagent volume 4744, and thereaction chamber 4732. As such, the first series of transfer pathways4793 and the second series of transfer pathways 4795 are configured toplace the reaction chamber 4732 in fluid communication with the firstdelivery pathway 4771 and the second delivery pathway 4773,respectively, and the reagent volume 4742 and the reagent volume 4744,respectively. In this manner, the contents of the reagent container 4780can be conveyed from the reagent container 4780 to the reaction chamber4732 via the reagent volume 4742, the first series of transfer pathways4793, and/or the first delivery pathway 4771. Similarly, the contents ofthe reagent container 4790 can be conveyed from the reagent container4790 to the reaction chamber 4732 via the reagent volume 4744, thesecond series of transfer pathways 4795, and/or the second deliverypathway 4773.

Moreover, although the housing 4741 is shown as having a first series oftransfer pathways 4793 and a second series of transfer pathways 4795, inother embodiments, the housing 4741 can have (or define) any suitablenumber of transfer pathways and/or series of transfer pathways. Althoughnot shown, in some embodiments, the first series of transfer pathways4793 (or a portion thereof) and the second series of transfer pathways4795 (or a portion thereof) can be in fluid communication with eachother. For example, in some embodiments, the first series of transferpathways 4793 and the second series of transfer pathways 4795 can be influid communication with each other via a transfer header pathway (notshown), wherein the transfer header pathway is in fluid communicationwith the reaction chamber 4732. In such embodiments, for example, thecontents of the first reagent container 4780 can communicate (e.g., mix)with the contents of the second reagent container 4790 before reachingthe reaction chamber 4732 or a portion thereof. Such an arrangement, insome embodiments, can promote mixing and/or minimize aeration, oversprayand/or undesirable turbulence of the contents from the reagent container4780 and/or the reagent container 4790.

Referring to FIGS. 11 and 16-18, the first actuator 4750 has a firstplunger portion 4754 disposed within the first reagent volume 4742, anda first engagement portion 4752. The second actuator 4760 (not shown inFIG. 16) has a second plunger portion 4756 disposed within the secondreagent volume 4744, and a second engagement portion 4753. Although theactuator shown in FIG. 16 is described herein with reference to actuator4750 for ease of explanation, it should be understood that any featuredescribed with reference to the first actuator 4750 can also, oralternatively, apply to the second actuator 4760, and vice-versa.

The first engagement portion 4752 of the first actuator 4750 isconfigured to be manipulated to move the first plunger portion 4754within the first reagent volume 4742 to deform the first reagentcontainer 4780. The second engagement portion 4753 of the secondactuator 4760 is configured to be manipulated to move the second plungerportion 4756 within the second reagent volume 4744 to deform the secondreagent container 4790. In this manner, movement of the plunger portion4754 can urge a frangible portion 4788 of the first reagent container4780 against the puncturer 4792 to pierce and/or rupture the frangibleportion 4788. Similarly, movement of the plunger portion 4756 can urge afrangible portion 4789 of the second reagent container 4790 against thepuncturer 4794 to pierce and/or rupture the frangible portion 4789. Theplunger portion 4754 of the actuator 4750 and a portion of the housing4741 can collectively define a seal to fluidically and/or opticallyisolate the reagent volume 4742 from a volume outside of the housing4741. Similarly, the plunger portion 4756 of the actuator 4760 and aportion of the housing 4741 can collectively define a seal tofluidically and/or optically isolate the reagent volume 4744 from avolume outside of the housing 4741.

Moreover, although the plunger portion 4754 shown in FIG. 16 has asubstantially planar surface for contacting the reagent container 4780,in other embodiments, the plunger portion 4754 can be any suitableshape, size, and/or configuration. For example, in some embodiments, theplunger portion 4754 can correspond to (e.g., share a similar shape,cooperatively function) the reagent container 4780 (e.g., the contactportion of the reagent container) and/or the puncturer 4792. Forexample, in some embodiments, the plunger portion 4754 can be curved(e.g., concave) so as to mate with a curved (e.g., concave) portion ofthe reagent container 4780. In this manner, the plunger portion 4754 andthe reagent container 4780 can collectively and/or cooperativelyfunction to limit dead volume. Moreover, such cooperation (e.g., mating)can promote repeatable delivery of the contents of the reagent container4780. Similarly, in some embodiments, for example, the plunger portion4754 can be curved so as to mate with a curved portion of the puncturer4792. In this manner, the plunger portion 4754 and the puncturer 4792can collectively and/or cooperatively function to limit dead volume.Moreover, such cooperation (e.g., mating) can promote repeatabledelivery of the contents of the reagent containers 4780.

As shown in FIG. 15, the reagent container 4780 has a sidewall 4786 anda frangible portion 4788, which together define an internal volume. Theinternal volume can be completely or partially filled with a reagentand/or substance, as described herein. In addition, the reagentcontainer 4780 has a skirt 4781 (referred to as a “first skirt”), acontact portion 4782 (referred to as a “first contact portion”), and afrangible portion 4788 (referred to as a “first frangible portion”). Theskirt 4781 surrounds at least a portion of the frangible portion 4788.In some embodiments, the sidewall 4786 can also be frangible. Thereagent container 4790 has a skirt 4791 (referred to as a “secondskirt”), a contact portion 4792 (referred to as a “second contactportion”), and a frangible portion 4789 (referred to as a “secondfrangible portion”). The second skirt 4791 surrounds at least a portionof the second frangible portion 4789. It should be noted that althoughthe reagent container shown in FIG. 15 is described with reference toreagent container 4780 for ease of explanation, any feature describedwith reference to reagent container 4780 can also, or alternatively,apply to reagent container 4790 and vice-versa.

The first skirt 4781 and/or the second skirt 4791 can be any suitablesize and/or shape, and can include any suitable surface design (e.g.,smooth, rough and/or the like). For example, in some embodiments, thefirst skirt 4781 and/or the second skirt 4791 can be sized and/or shapedto correspond to a portion of the housing 4741. The first contactportion 4782 of the reagent container 4780 and/or the second contactportion 4792 of the reagent container 4790 can be any suitable sizeand/or shape. For example, in some embodiments, the first contactportion 4782 and/or the second contact portion 4792 can be sized and/orshaped to correspond to the first actuator 4750 and/or the secondactuator 4760, respectively. For example, in such embodiments, the firstcontact portion 4782 and/or the second contact portion 4792 can includea concave portion, and the first actuator 4750 and/or the secondactuator 4760 can be sized and/or shaped to correspond to the concaveportion of the first contact portion 4782 and/or the concave portion ofthe second contact portion 4792, respectively. In this manner, thereagent container 4780 and/or the reagent container 4790 can beconfigured to promote substantially complete dispensation of theirrespective contents (e.g., reagents, substances, etc.), and/or promote apreferred pathway for the contents to travel from the reagent container4780 and/or the reagent container 4790 when the reagent container 4780and/or the reagent container 4790 are pierced.

The reagent container 4780 is shaped and sized to be disposedsubstantially inside the first reagent volume 4742. The reagentcontainer 4790 is shaped and sized to be disposed substantially insidethe first reagent volume 4744. As best illustrated in FIGS. 17 and 18,the reagent container 4780 can be maintained in a desired position by aninterference fit between the first skirt 4781 and a portion of thehousing 4741. Similarly, the reagent container 4790 can be maintained ina desired position by an interference fit between the second skirt 4791and a portion of the housing 4741. In this manner, a desired position ofthe reagent container 4780 and/or the reagent container 4790 can besubstantially maintained relative to the housing 4741 during use.

Although the container assembly 4700 is not shown as including a lockmember, in some embodiments, the container assembly 4700 can include alock member similar to the lock member 2772 shown and described abovewith reference to FIG. 5. In such embodiments, the reagent container4780 can be maintained in a desired position by the lock member (notshown) and by an interference fit between the first skirt 4781 and aportion of the housing 4741 and/or a portion of the lock member.Similarly, in such embodiments, the reagent container 4790 can bemaintained in a desired position by the lock member (not shown) and byan interference fit between the second skirt 4791 and a portion of thehousing 4741 and/or a portion of the lock member.

The reagent container 4780 and/or the reagent container 4790 can haveany suitable size and/or volume. For example, in some embodiments, thereagent container 4780 and/or the reagent container 4790 can have aninternal volume of about 400 μL when in the expanded configuration. Insuch embodiments, the reagent container 4780 and/or the reagentcontainer 4790 can initially contain about 300 μL to about 350 μL (andmore particularly, about 325 μL) of any of the reagents describedherein. Thus, when the reagent container 4780 and/or the reagentcontainer 4790 are in their respective expanded configurations, theyhave a fill percentage of about 75 percent to about 88 percent. Thereagent container 4780 and/or the reagent container 4790 are configured,along with their respective plungers and portions of the housing, suchthat when in their respective collapsed configurations, the dispensedvolume is about 250 μL to about 300 μL (and more particularly, about 285μL). Similarly stated, when the reagent container 4780 and/or thereagent container 4790 are in their respective collapsed configurations,they have a dispensation percentage of between about 76 percent andabout 92 percent.

The first reagent container 4780 and the second reagent container 4790can be completely or partially filled with any suitable reagent orsubstance. In some embodiments, the first reagent container 4780 and thesecond reagent container 4790 can include the same contents (e.g., thesame reagent). In other embodiments, the first reagent container 4780and the second reagent container 4790 can include dissimilar contents(e.g., the first reagent container 4780 contains a first reagent and thesecond reagent container contains a second reagent different than thefirst reagent). In some embodiments, for example, the reagent container4780 and/or the reagent container 4790 can contain transductionparticles that include an engineered nucleic acid formulated to causethe target cell (e.g., bacteria) to produce one or more reportermolecules. In some embodiments, the reagent container 4780 and/or thereagent container 4790 can contain one or more transduction particlesengineered to be incapable of replication (e.g., lytic replication,lysogenic replication). For example, in some embodiments, the reagentcontainer 4780 and/or the reagent container 4790 can contain any of thetransduction particles described herein and in U.S. ProvisionalApplication Nos. 61/983,765, entitled “Reagent Cartridge for Detectionof Cells,” filed Apr. 24, 2014; 61/779,177, entitled “Non-ReplicativeTransduction Particles and Transduction Particle-Based ReporterSystems,” filed Mar. 13, 2013; 61/939,126, entitled “Systems and Methodsfor Packaging Nucleic Acid Molecules into Non-Replicative TransductionParticles and Their Use as Cellular Reporters,” filed Feb. 12, 2014; and61/897,040, entitled “Transcript Detection Systems and Methods,” filedOct. 29, 2013, and International Patent Application No.PCT/US2014/026536, entitled “Non-Replicative Transduction Particles andTransduction Particle-Based Reporter Systems,” filed Mar. 13, 2014, eachof which is incorporated herein by reference in its entirety.

In some embodiments, the reagent container 4780 and/or the reagentcontainer 4790 can contain a reagent formulated to react with one ormore reporter molecules to generate and/or enhance production of asignal. For another example, the reagent container 4780 and/or thereagent container 4790 can include a luciferase substrate, such astridecanal, that can interact with a reporter molecule (e.g.,luciferase), to produce a measurable signal, e.g., via a luminescencereaction. For yet another example, in some embodiments, the reagentcontainer 4780 and/or the reagent container 4790 can include a nutrient,an antibiotic (e.g., Beta-lactams, extended-spectrum beta-lactams,Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, any generation ofCephalosporins, Glycopeptides, Lincosamides, Lipopeptide, Macrolides,Monobactams, Nitrofurans, Oxazolidonones, Penicillins, Polypeptides,Quinolones, Fluoroquinolones, Sulfonamides, Tetracyclines, mycobacterialantibiotics, Chloramphenicol, Mupirocin), a lysis reagent, a sterilizingreagent, a colorant and/or the like.

The reagent container 4780 and/or the reagent container 4790 can beconstructed from any suitable materials having any suitable dimensions.The thickness of the sidewall of the reagent container 4780 and/or thereagent container 4790 can be, for example, between about 0.010 inchesand 0.020 inches. Moreover, the reagent container 4780 and/or thereagent container 4790 can be constructed from materials that aresubstantially impermeable to and/or substantially chemically inert fromthe substance(s) contained therein, e.g., transduction particle,substrate, antibiotics, buffers, surfactants, or any other reagent thatcan be used with the detection assay. At least a portion of the reagentcontainer 4780 (e.g., the frangible portion 4788) and/or at least aportion of the reagent container 4790 (e.g., the frangible portion 4789)can be constructed from a material (e.g., polymer film, such as any formof polypropylene) having certain temperature characteristics such thatthe desired properties and integrity are maintained over a certaintemperature. For example, in some instances, it can be desirable tostore the reagent container 4780 and/or the reagent container 4790containing reagent and/or substrate in a refrigerated condition. In someembodiments, a portion of the reagent container 4780 and/or a portion ofthe reagent container 4790 can be constructed from bi-axially orientedpolypropylene (BOP). In some embodiments, a portion of the reagentcontainer 4780 and/or a portion of the reagent container 4790 can beconstructed from aluminum. In some embodiments, a portion of the reagentcontainer 4780 and/or a portion of the reagent container 4790 can beconstructed from polyvinyl chloride (PVC), ethylene vinyl alcohol(EVOH), polyethylene (PE), polychlorotrifluoroethene (PCTFE or PTFCE), apharmaceutical-grade copolymer, cyclic olefin copolymer film, Tekniflex,COC P12P, PCTFE, film lamination, and/or Tekniflex VA10200.

For example, in some embodiments, the reagent container 4780 and/or thereagent container 4790 can be constructed from PVC having a laminate ofpolyethylene EVOH on the interior surface of the sidewalls. In thismanner, the laminate can function as an oxygen barrier to preserve thereagents contained within the reagent container 4780 and/or the reagentcontainer 4790. In some embodiments, an outer surface can include aPCTFE coating to function as a moisture barrier. In some embodiments,the frangible portion 4788 and/or the frangible portion 4789 are weldsealed to the sidewalls. Moreover, in some embodiments, the frangibleportion 4788 and/or the frangible portion 4789 can be devoid of thecoatings to provide sufficient “puncturability” or minimum rupturestrength for repeatable operation. In other embodiments, the frangibleportion 4788 and/or the frangible portion 4789 can include a lacquercoating.

The reaction chamber 4732 can be removably coupled to the housing 4741.As shown, the reaction chamber 4732 is threadedly coupled to the housing4741. In other embodiments, however, the reaction chamber 4732 can forman interference fit to couple the reaction chamber 4732 to the housing4741. The reaction chamber 4732 includes a sidewall portion 4734 and adistal portion (including a bottom surface) 4736, and can be anysuitable chamber for containing a clinical sample (e.g., a patientsample) in a manner that permits the monitoring, identification, and/ordetection of a target cell (e.g., bacteria) within the sample. In someembodiments, at least a portion of the reaction chamber 4732, such asthe distal portion 4736, can be substantially transparent, for example,to allow viewing, and/or optical monitoring of the contents containedtherein. In some embodiments, a portion of the reaction chamber 4732(e.g., a distal portion) can be substantially transparent while theremainder of the reaction chamber 4732 can be substantially opaque. Inthis manner, the reaction chamber 4732 can be configured to convey lightthrough the substantially transparent portion of the reaction chamber4732, but block light at the substantially opaque portion of thereaction chamber 4732. In some embodiments, the sidewall portion 4734 ofthe reaction chamber 4732 can include a coating to allow for optimaltransmission of light through the distal portion 4736 of the reactionchamber 4732. In some embodiments, the coating can be any suitablematerial configured to block and/or reflect light, for example, a label.In particular, in some embodiments, the label can be a white label toreflect light. Moreover, in some embodiments, the distal portion 4736 ofthe reaction chamber 4732 can be polished to promote optimaltransmission of light therethrough.

FIGS. 17 and 18 show a cross-sectional side view of container assembly4700 in a first configuration (FIG. 17) and a second configuration (FIG.18), respectively. As shown, the distal portion 4736 of the reactionchamber 4732 includes a substantially flat bottom surface. The flatbottom surface promotes substantially uniform delivery of lighttherethrough. Specifically, in use, light can be transmitted through thedistal portion 4736 substantially uniformly to a detector. Similarlystated, this arrangement allows a “bottom read” of the containerassembly 4732 by the detector (e.g., any detector described herein andin the '461 Application). Moreover, in use, such a substantially flatsurface at the distal portion 4736 can result in the container assembly4700 being placed consistently closer to and/or in contact with anoptical detection window in an instrument. In this manner, such aconfiguration can minimize the distance in the signal path betweensignal production and signal detection and/or minimize an interfacebetween mismatched dialectic mediums in the signal path, both of whichcan contribute to loss in signal reaching the sensor, e.g., due to lightscattering and/or light refraction. Moreover, in some embodiments, forexample, the flat surface can be configured to contact the opticaldetection window. Furthermore, as shown in FIGS. 17 and 18, the sidewallportion 4734 of reaction chamber 4732 is tapered. Similarly stated, asurface of the sidewall portion 4734 is nonparallel to a longitudinalcenterline defined by the reaction chamber 4732. The taperedconfiguration promotes flow of contents from the reagent container 4780and/or the reagent container 4790 along the sidewall portion 4734. Assuch, turbulence, splash, the production of bubbles, aeration, and/orthe like, of the contents can be limited, and subsequent opticalreadings can be more accurate than if the sample contains such bubbles,aeration or the like. In particular, an exit portion of the firstdelivery pathway 4771 and an exit portion of the second delivery pathway4773 each define an exit axis (the axis EE and the axis FF,respectively) that intersects the sidewall portion 4734 of the reactionchamber 4732, as shown in FIG. 17. Thus, in use, contents from thereagent container 4780 and/or the reagent container 4790 can flow fromthe first delivery pathway 4771 and/or the second delivery pathway 4773,respectively, to the sidewall portion 4734 along their respective exitaxis. Moreover, as shown, the intersection of each exit axis (i.e., axisEE and axis FF) and the sidewall portion 4734 occurs above the expectedfill line 4738 (or nominal fill line), thus preventing and/or limitingsplashing, turbulence or the like, of the contents as the contents movefrom the reagent container 4780 and/or the reagent container 4790 to thereaction chamber 4732. Although the sidewall portion 4734 of reactionchamber 4732 is shown as tapered such that each exit axis intercepts thesidewall portion 4734 at a top-half portion of the reaction chamber4732, in other embodiments, the sidewall portion 4734 can be tapered (orangled) at any suitable degree. In some embodiments, the sidewallportion 4734 can be tapered (with respect to the longitudinalcenterline) by about one degree. In other embodiments, the sidewallportion 4734 can be tapered (with respect to the longitudinalcenterline) by less than 5 degrees. Similarly, although the nominal fillline 4738 is illustrated to be located near a middle portion of thereaction chamber 4736, in other embodiments, the nominal fill line canbe located at any suitable level (e.g., as a function an angleassociated with the sidewall portion 4734 and/or either exit axis).

The reaction chamber 4732 can be constructed from any suitable material,for example, glass, plastic (e.g., polypropylene), acrylic, etc. In someembodiments, the reaction chamber 4732 can be gamma sterilizable. Insome embodiments, the reaction chamber 4732 can be a commerciallyavailable container, for example a centrifuge tube, an Eppendorf® tube,a glass vial, flat-bottomed vial/tube, round bottomed vial/tube, or anyother suitable container.

As shown in FIG. 17, the container assembly 4700 is in a firstconfiguration. In the first configuration, the first actuator 4750 andthe second actuator 4760 are positioned such that the reagent container4780 and the reagent container 4790 disposed within the housing 4741 aresubstantially undeformed. Similarly stated, the first actuator 4750 andthe second actuator 4760 are positioned such that they do not causepuncturer 4752 and puncturer 4794 to pierce the reagent container 4780and the reagent container 4790, respectively. Thus, the containerassembly 4700 is in a “ready” state when in the first configuration. Insome embodiments, the container assembly 4700 can include a safetymechanism (not shown) to prevent and/or limit movement of the firstactuator 4750 and/or the second actuator 4760 relative to the housing4741 until desired by the operator.

To actuate the container assembly 4700, a force is applied to theengagement portion 4752 of the first actuator 4750, and a force isapplied to the engagement portion 4753 of the actuator 4760, thuscausing the first actuator 4750 and the second actuator 4760 to move asshown by the arrow GG and HH, respectively, in FIG. 17. The forces canbe applied by any suitable instrument, such as those shown and describedin the '461 application. The forces can be applied substantiallysimultaneously or at different times, in accordance with the desiredassay.

More particularly, the first actuator 4750 is manipulated (e.g., at thefirst engagement portion 4752) to move the first plunger portion 4754within the housing 4741 such that the first plunger portion 4754 engagesthe contact portion 4782 of the reagent container 4780 to partiallydeform the reagent container 4780 from the first configuration to thesecond configuration. As the first plunger portion 4754 engages thereagent container 4780, the first puncturer 4792 pierces a portion ofthe reagent container 4780 (e.g., the frangible portion 4788) to conveyreagent from the reagent container 4780 into the first reagent volume4742, the first delivery portion 4770, and/or the reaction chamber 4732.Similarly, the second actuator 4760 is manipulated (e.g., at the secondengagement portion 4753) to move the second plunger portion 4756 withinthe housing 4741 such that the second plunger portion 4756 engages thesecond contact portion 4784 of the reagent container 4790 to partiallydeform the reagent container 4790 from the first configuration to thesecond configuration. As the second plunger portion 4756 engages thereagent container 4790, the second puncturer 4794 pierces a portion ofthe reagent container 4790 (e.g., the frangible portion 4789) to conveyreagent from the reagent container 4790 into the second reagent volume4744, the second delivery portion 4772, and/or the reaction chamber4732.

As shown in FIG. 18, and in greater detail in FIG. 19, the containerassembly 4700 is in a second configuration. In the second configuration,the first actuator 4750 and the second actuator 4760 are positioned suchthat the reagent container 4780 and the reagent container 4790 aresubstantially deformed and/or collapsed. Similarly stated, the firstactuator 4750 and the second actuator 4760 are positioned such that atleast portions of the respective forces are transferred to the firstreagent container 4780 and the second reagent container 4790,respectively. In such a configuration, as shown, the first puncturer4792 has pierced the reagent container 4780 such that a desired amountof the contents of the reagent container 4780 have substantially exitedthe reagent container 4780, and entered the first delivery portion 4770and/or the reaction chamber 4732, as shown by the arrow II. Similarly,the second puncturer 4794 has pierced the reagent container 4790 suchthat a desired amount of the contents of the reagent container 4790 havesubstantially exited the reagent container 4790, and entered the seconddelivery portion 4772 and/or the reaction chamber 4732, as shown by thearrow JJ.

When the reagent container 4780 and/or the reagent container 4790 aredeformed, a desired amount of its contents are conveyed into thereaction chamber 4732 in a manner such that “dead volume” is limitedand/or substantially eliminated. As used herein the “dead volume” is thevolume of reagent that is dispensed from the reagent container 4780and/or the reagent container 4790 but that is not conveyed into reactionchamber 4732. The dead volume can include, for example, the volume ofthe delivery pathways and the transfer pathways. In some embodiments,the reagent container 4780 and/or the reagent container 4790 can beconfigured to limit the dead volume therein when the assembly 4700 isactuated. For example, in some embodiments, the contact portion 4782and/or the contact portion 4784 can be configured, along with thecorresponding engagement portions of the actuator 4750 and actuator4760, respectively, to deform in a controlled manner that reduces thedead volume. In this manner, the reagent container 4780 and/or thereagent container 4790 can be configured to promote a consistent and/orrepeatable dispensation of their contents (e.g., reagents)

In some embodiments, the cap assembly (i.e., the reagent container 4780and/or the reagent container 4790 along with their respective plungersand portions of the housing) is configured such that the “dead volume”is between about 30 μL and about 50 μL. In some embodiments, the capassembly is configured such that the “dead volume” about 40 μL±9 μL. Bylimiting the part-to-part variation in the dead volume, the accuracy ofreagent delivery, and thus, the accuracy of the assay, can be improved.In some embodiments, for example, the cap assembly is configured suchthat the dispensed volume is about 285 μL with a coefficient ofvariation of about three percent.

As described herein, in some embodiments, a container assembly (e.g.,container assembly 4700 or any other container assembly describedherein) can contain a patient sample that potentially contains a cell(e.g., a bacteria) to be detected using the methods, instruments and/orany of the components described herein and in the '461 application,which is incorporated herein by reference in its entirety. The samplecan be a human sample (e.g., a nasal swab, mucosal swab, saliva sample,blood sample, urine sample, fecal sample, tissue biopsy, bone marrowand/or cerebrospinal fluid), veterinary sample, food sample, plantsample, and/or environmental sample. In some embodiments, the sample canbe a crude, raw, or otherwise substantially unprocessed sample.

In some embodiments, a kit can be provided and/or used to perform suchmethods. For example, FIG. 20 illustrates a kit 4000, according to anembodiment. As shown, the kit 4000 includes a transport containerassembly 4010, a transfer member 4030, and the container assembly 4700.Although shown as including the container assembly 4700, in otherembodiments, a kit can include any of the container assemblies and/orcap assemblies as shown and described herein.

The transport container assembly 4010 (also referred to as the“collection assembly”) includes a sample collector 4020, a transport cap4012 and a transport chamber 4014. The transport cap 4012 is removablycoupleable to the transport chamber 4014 to form a substantiallyfluid-tight seal. For example, in some embodiments, the transport cap4012 can be threadedly coupled to the transport chamber 4014. In otherembodiments, the transport cap 4012 and the transport chamber 4014 canform an interference fit, press fit, snap fit, and/or any other suitablefit to couple the transport cap 4012 to the transport chamber 4014.

The transport chamber 4014 can be any suitable size and/or shape, andcan be constructed from any suitable material. The transport chamber4014 defines a transport volume 4016 within which the sample can bedisposed. In some embodiments, the transport chamber 4014 can includewithin the transport volume 4016 a transport media, solution and/orreagent (not identified in FIG. 20). The transport media can include,for example, a bacterial nutrient media, organism selective media,buffers, surfactants or any other component to facilitate growth and/oroptimize the health of the patient sample (e.g., target bacteria),production of reporter molecules within the target bacteria, detectionof bacteria and/or the like. In some embodiments, the transport mediacan include, for example, a bacterial nutrient and/or growth media(e.g., undefined medium, defined medium, differential medium, minimalmedia, selective media, etc.) to enable bacteria to grow and multiply, abuffer to maintain pH (e.g., Amies, PBS, HEPES, TRIS, TAPSO, Bicine,MES, MOPS, Tricine, PIPES, SSC, succinic acid, etc.) and/or a surfactant(e.g., Tween 20, Tween 80, TritonX, X-114, CHAPS, DOC, NP-40 CTAB, SDS,etc.). In some embodiments, the transport media or transport compositioncan be predisposed in the internal volume 4016 or it can be added afterthe sample is conveyed into the container. In some embodiments, thetransport media can be predisposed in the transport chamber 4014, butcan be selectively maintained in isolation from the sample, e.g., in aseparate compartment (not shown) within the transport chamber 4014. Forexample, in some embodiments, the transport media can be stored in thetransport cap 4012 such that the solution can be communicated to thepatient sample on demand and/or in a closed-system environment.

In some embodiments, the transport media, reagent and/or composition canbe tailored to enhance growth, shorten lag phase, sustain, and/or attacka particular target cell, e.g., bacterium. In some embodiments, specificversions of the solution can be employed for specific target cellsand/or samples. For example, a first preparation of the solution can betailored for nasal swab samples containing MRSA, a second preparation ofthe solution can be tailored for urine samples containing E. coli, athird preparation of the solution can be tailored for stool samplescontaining C. difficile, and the like.

The sample collector 4020 includes a shaft portion 4022 and a collectionportion 4024. The shaft portion 4022 is configured to be coupled (e.g.,removably or substantially permanently) to the transport cap 4012. Forexample, in some embodiments, the shaft portion 4022 of the samplecollector 4020 can be removably coupled to the transport cap 4012 afterthe sample collector 4020 has been used to collect a patient sample. Inother embodiments, for example, the shaft portion 4022 can remaincoupled to the transport cap 4012 while the sample collector 4020 isused to collect a patient sample. In this manner, in some embodiments, auser can handle the sample collector using the transport cap, while inother embodiments, a user can handle the sample collector using theshaft portion 4022.

The sample collector 4020 can have any suitable configuration andmaterial for collecting a patient sample. For example, in someembodiments, the sample collector 4020 can be a swab (e.g., a woundswab, a flock swab, a foam swab, etc.). Moreover, in some embodiments,the sample collector 4020 (and more specifically, the collection portion4024) can be configured to release at least a portion of the patientsample into the transport media. In this manner, the sample collector4020 can release a patient sample into the transport chamber 4014 forlater transfer into the container assembly 4700, as described herein.

Accordingly, in some embodiments, the sample collector 4020 isconfigured to and/or constructed from materials formulated to maximizethe sample collection efficiency and the efficiency of releasing thecollected sample into the container assembly 4700 and/or reactionchamber 4732. As supported by the examples provided herein, in somesituations, it has been determined that foam swabs perform better thanwound and/or flocked swabs in a MRSA screen assay when the swab istransported in an assay media (e.g., via a transport chamber 4014, withan assay bead and cap 4012). In yet other instances, it has beendetermined that foam and/or flocked swabs perform relatively similarly,and both better than wound swabs, when the swab is transported alongwith the sample within the transport chamber 4014. In some instances, ithas been determined that wound swabs (e.g., wound Rayon swabs) and/orDacron swabs, although traditionally used in various sampling methods,are poor at releasing the patient sample (e.g., bacteria) into an assay.In other instances, it has been determined that flocked swabs performbetter than traditional swabs (e.g., wound swabs) at releasing thepatient sample but, in some instances, perform poorly in the assay. Sucha poor performance, in some instances, can be mitigated by performingcertain transport methods. Such transport methods can includetransferring the patient sample from the patient swab to a reactionchamber (e.g., the reaction chamber 4732) without placing the patientswap in the transport chamber 4014 (e.g., transfer the patient samplevia a transfer tool). In this manner, in some instances, it has beendetermined that flocked swabs can perform well in the assay. In furtherinstances, it has been determined that foam swabs, while not typicallyused in bacterial assays, release the bacteria as well as flocked swabsand perform well in the assay.

The transfer tool 4030 can be any suitable tool used to transfer thetransport media and/or sample from the transport chamber 4014 to areaction chamber (e.g., reaction chamber 4732). For example, in someembodiments, the transfer tool 4030 can be a pipette. In otherembodiments, for example, the transfer tool 4030 can be a swab. In yetfurther embodiments, for example, the transfer tool 4030 can be asyringe. The transfer tool 4030 can be any suitable size and shape, andcan be constructed from any suitable material.

FIG. 21 is a flow diagram illustrating a method 4200 for collecting,transporting, and testing a patient sample using the kit 4000 or anyother devices shown and described herein. As shown at 4210, a patientsample is collected from a patient (e.g., via a nostril of the patient)using sample collector 4020. At 4220, the sample collector 4020 isplaced into transport container assembly 4010 such that the patientsample is disposed within the transport volume 4016 of the transportchamber 4014. In particular, the sample collector 4020 is deposed withinthe transport volume 4016 such that the collection portion 4024 isdisposed within any transport media within the transport chamber 4014.The transport cap 4012 is then coupled to the transport chamber 401,leaving the sample collector 4020 within the transport chamber 4014. Inthis manner, the patient sample can be communicated from the samplecollector 4020 (e.g., from the collection portion 2024) to the transportvolume 4016 and/or the transport media of the transport chamber 4014. Insome embodiments, operations 4210 and 4220 can occur at a point ofcollection, such as, for example, a nurse station.

At 4230, at least a portion of the patient sample (e.g., thatpotentially includes the target bacteria) and the transport media can becommunicated from the transport container assembly 4010 and/or thesample collector 4020 via transfer tool 4030 (e.g., a pipette) into thereaction chamber (e.g., the chamber 4732). In some instances, thetransport cap 4012 is first separated from the transport chamber 4014.As such, the transfer tool 4030 can access a least a portion of thepatient sample disposed within the transport chamber 4014. In thismanner, a target bacterium is transferred from via transfer tool 4030into the reaction chamber 4732 of container assembly 4700. Althoughshown as container assembly 4700, any suitable container assembly can beused (e.g., container assembly 1700, 2700, 3700, etc.).

At 4250, the container assembly 4700 containing target cells is disposedwithin a detection instrument (e.g., any instrument described herein andin the '461 application, which is incorporated herein by reference inits entirety). The container assembly 4700 is then subjected to themethods of detection described herein and in the '461 application.

In some embodiments, a method can involve using a sample collectorformulated to maximize the sample collection efficiency and/or theefficiency of releasing the collected sample into a container assembly.For example, FIG. 22 is a flow chart of a method 200 according to anembodiment. As shown in FIG. 22, the method 200 includes receiving acontainer containing a swab and a transport media, at 205. The swab canbe similar to the sample collectors described herein, and includes ashaft having a collection portion constructed from non-wound material.In some embodiments, the collection portion can be constructed from amaterial that is electrostatically assembled or otherwise flocked (i.e.,a flocked swab) or a foam material, such as an open-cell foam-tippedswab. The transport media includes a sample released from the collectionportion.

At 210, the transport media and the sample are transferred into areaction chamber. The reaction chamber can be any reaction chamberdescribed herein, such as the reaction chamber 4732. The transport mediaand the sample can be transferred via any suitable mechanism, such asvia the transfer member 4030 (e.g., a pipette).

The transport media and one or more transduction particles associatedwith a target cell are mixed in the reaction chamber, at 215. Thetransduction particles can be any transduction particles describedherein, and are engineered to include a nucleic acid molecule formulatedto cause the target cell to produce one or more reporter molecules. Insome embodiments, the one or more transduction particles can benon-replicative. In some embodiments, the one or more transductionparticles can be devoid of a reporter molecule of the one or morereporter molecules. In yet further embodiments, the reporter moleculefrom the one or more reporter molecules can include one or more of abacterial luciferase, an eukaryotic luciferase, a fluorescent protein,an enzyme suitable for colorimetric detection, a protein suitable forimmunodetection, a peptide suitable for immunodetection or a nucleicacid that function as an aptamer or that exhibits enzymatic activity.

The transduction particles can be added to and/or mixed within thereaction chamber by any suitable mechanism. In some embodiments, thetransduction particles can be included in the reagent container 4780and/or the reagent container 4790 within the housing 4741 (or capassembly) as described herein. In such embodiments, the transductionparticles can be added to and/or mixed within the reaction chamber(e.g., the chamber 4732) by the application of a force to an actuator(e.g., the actuator 4750 and/or the actuator 4760), which thereby causesthe transduction particles to be conveyed from the reagent containerinto the reaction chamber as described herein. In some embodiments, thetransduction particles can be conveyed such that the dead volume withinthe cap assembly is between about 30 μL and about 50 μL. In someembodiments, the transduction particles can be conveyed such that the“dead volume” about 40 μL±9 μL. In some embodiments, the transductionparticles can be conveyed such that the dispensed volume is about 285 μLwith a coefficient of variation of about three percent. By limiting thedead volume and/or the part-to-part variation in the dead volume, theaccuracy of delivery, and thus, the accuracy of the assay, can beimproved.

In some embodiments, the transduction particles can be conveyed into thereaction chamber in a manner that reduces the turbulence generatedtherein. For example, in some embodiments, the transduction particlescan be conveyed such that they impinge and/or contact the sidewall ofthe reaction chamber as described herein. In other embodiments, thetransduction particles can be conveyed at a velocity and/or flow rate topromote mixing and/or reduce turbulence. For example, in someembodiments, the mixing of the transduction particles includes conveyingthe transduction particles into the reaction chamber by moving theactuator (e.g., the actuator 4750) linearly at a rate of between about63 mm per second and about 81 mm per second. In some embodiments, themixing of the transduction particles includes conveying the transductionparticles into the reaction chamber by moving the actuator (e.g., theactuator 4750) linearly at a rate of about 72 mm per second.

In some embodiments, the reaction chamber can contain a reagent (e.g.,in dried form including tablet form, and/or including an antibiotic, asdescribed herein) formulated to mix with the transport media. Theantibiotics can be selected and/or formulated to kill other non-targetedbacterial strains, for example, non-drug resistant strains, so that onlythe drug resistant strain survives. In this manner, the reportermolecules produced are necessarily produced by the remaining, targetedbacterial strains. In some embodiments, the antibiotic/series ofantibiotics can be predisposed in the reaction chamber (for example, inthe transport media, in a freeze-dried and/or lyophilized form or anyother suitable form). In other embodiments, the antibiotic/series ofantibiotics can be disposed in a separate compartment (e.g., in areagent container, such as the reagent container 4790), and can becommunicated into the sample solution on demand or at a predeterminedtime.

The mixture of the transport media and the one or more transductionparticles is maintained at a temperature of at least 20 degrees Celsiusfor a period of about eight hours or less to express the one or morereporter molecules when the target cell is present in the sample, at220. In some embodiments, the mixture can be maintained at about 37degrees Celsius for about four hours. In yet other embodiments, themixture can be maintained for about three hours or less, or about 2hours or less. In yet other embodiments, the mixture can be maintainedand at any suitable temperature, e.g., between the range of about 20degrees Celsius and about 37 degrees Celsius.

A signal associated with a quantity of the one or more reportermolecules is received, at 225. The signal can be any suitable signalthat is produced by certain reporter molecules, such as for example, anoptical signal produced by a flash luminescence reaction. In someembodiments, the signal is associated with the quantity of reportermolecules within the sample. In some embodiments, the magnitude of thesignal is independent from the quantity of the transduction particlesabove a predetermined quantity. Similarly stated, in some embodiments,the strength of the signal is substantially independent from thequantity of the transduction particles.

Although kit 4000 includes transport container assembly 4010, transfertool 4030, and container assembly 4700, in other embodiments, a kit caninclude additional components and/or be devoid of any such components.For example, in some embodiments, a kit can be devoid of a transfertool. In some embodiments, a kit can be devoid of a transfer tool and atransport chamber. In such embodiments, for example, a kit can include atransport cap (e.g., transport cap 4012, a sample collector (e.g.,sample collector 4020), and a container assembly (e.g., containerassembly 4700 or any other container assembly described herein). In thismanner, a patient sample collected with and disposed on a samplecollector can be placed within a reaction chamber (e.g., reactionchamber 4732 or any other reaction chamber described herein). In suchembodiments, for example, the transport cap can be removably coupled tothe reaction chamber. In this manner, the transport cap can be removedfrom the reaction chamber, and the reaction chamber can be removablycoupled to a housing (e.g., housing 4741 or any other housing or capassembly described herein). In yet further embodiments, a kit caninclude a transport cap (e.g., transport cap 4012), a sample collector(e.g., sample collector 4020), and a reaction chamber (e.g., reactionchamber 4732 or any other reaction chamber described herein).

Although FIG. 21 illustrates a method including transport containerassembly 4010 and a separate reaction chamber, in other embodiments, apatient sample can be communicated from the sample collector 4020directly to a reaction chamber (e.g., a reaction chamber 4732) withoutfirst being transferred to a transport container. In such embodiments,the patient sample can be communicated from the sample collector 4020directly to the reaction chamber 4732, without the use of transfer tool4030. In such embodiments, the patient sample can be collected at acollection site via sample collector 4020 (e.g., similar to operation4210 described above). Next, the sample collector 4020 can becommunicated to reaction chamber 4732, or any other reaction chamberdisclosed herein. In this manner, the patient sample is communicatedfrom the patient to the reaction chamber 4732. Once at least a portionof the patient sample and/or the sample collector 4020 are disposedwithin the reaction chamber 4732, transport cap 4012 can be removablycoupled to the reaction chamber 4732. As such, the patient sample can becollectively contained and/or protected by the transport cap 4012, thereaction chamber 4732, and/or the sample collector 4020. In this manner,the patient sample can be stored, and/or transferred securely from thecollection site to a testing and/or detection site. When desired (e.g.,in preparation for target cell detection), the transport cap 4012 can beremoved from the reaction chamber 4732, and the housing 4741 (or capassembly) or any other housing described herein, can be removablycoupled to the reaction chamber 4732 to complete the assay.

In some embodiments, the transport chamber 4014 and/or reaction chamber4732 can contain a reagent or other composition formulated to mix withthe sample to form an assay media or solution. Such reagent can beincluded within and/or a portion of the transport media, oralternatively, can be a separate composition. For example in someembodiments, the transport chamber 4014 and/or the reaction chamber 4732can include a lyophilized tablet that is maintained separately from thetransport media, and is mixed with the sample to form an assay media.For example, in some embodiments, the transport chamber 4014 and/or thereaction chamber 4732 can contain antibiotics (e.g. cefoxitin,oxacillin, cefotetan, amoxycillin, penicillin, erythromycin,azythromycin, cephalosporins, carbapenems, aminoglycosides,sulfonamides, quinolones, oxazolidinones, etc.). The inclusion ofantibiotics can kill or otherwise prevent the expression and/orgeneration of a signal from reporter molecule from all drug-susceptiblebacteria, e.g., in a bacteria cell viability and/or susceptibility assayof the types shown and described herein. The antibiotics can be selectedand/or formulated to kill other non-targeted bacterial strains, forexample, non-drug resistant strains, so that only the drug resistantstrain survives. In this manner, the reporter molecules produced arenecessarily produced by the remaining, targeted bacterial strains. Insome embodiments, the antibiotic/series of antibiotics can bepredisposed in the container (for example, in the solution, in afreeze-dried and/or lyophilized form or any other suitable form). Inother embodiments, the antibiotic/series of antibiotics can be disposedin a separate compartment (e.g., in the body or cap of the containerassembly), and can be communicated into the sample solution on demand orat a predetermined time.

In some embodiments, the reaction chamber can include colorant (e.g., adye) along with any reagents disposed therein. Such dye can be used, forexample, as a “process control” to ensure that the contents of thecontainer (e.g., the reagents) were not disrupted and/or emptied beforethe sample was placed therein. In this manner, if during use, aninstrument senses color in the sample mixture, a signal can be sent toindicate and/or confirm that the dried reagent substance was actuallywithin the container. If no color is identified and/or detected, forexample, the instrument can send an error signal indicating that thedesired reagents were not, in fact, within the container during testing.

In particular, FIG. 23 is a flow chart of a method 100 according to anembodiment. As shown in FIG. 23, the method 100 includes disposing asample into a reaction chamber (any of the reaction chambers describedherein, e.g., reaction chamber 4732), at 105. The reaction chamber ispackaged to contain a reagent (e.g., a dried reagent, a lyophilizedreagent) formulated to mix with the sample to form an assay media. Insome embodiments, the reagent could be in pellet form. In otherembodiments, the reagent can be dried in the tube (e.g., adhered to aninner surface of the reaction chamber 4732). Moreover, in someembodiments, the reagent can include an antibiotic and a colorant. Insuch embodiments, the antibiotic can be formulated to suppressproduction of the one or more reporter molecules in the portion of thecell phenotype, as described herein. In other embodiments, the reagentcan include a substance formulated to suppress the transfer and/orconveyance of a detectable signal.

The sample in the reaction chamber is mixed with one or moretransduction particles associated with a cell phenotype, at 110. Thetransduction particles can be contained in the reagent container 4780and/or the reagent container 4790 and can be introduced into the sampleas described herein. The one or more transduction particles areengineered to include a nucleic acid molecule formulated to cause thecell phenotype to produce one or more reporter molecules capable ofgenerating and/or producing a detectable signal. In some embodiments,the detectable signal can be an optical signal produced by a flashluminescence reaction. In some embodiments, the transduction particlescan be engineered to be incapable of lytic and/or lysogenic replication.In some embodiments, the one or more transduction particles can bederived from a bacteriophage. The reagent is formulated to suppress thedetectable signal, either by suppressing production of compositions thatproduce the signal or by suppressing the conveyance and/or transmissionof the signal. In some embodiments, for example, the reagent isformulated to suppress production of the one or more reporter moleculesin at least a portion of the cell phenotype.

The transduction particles can be added to and/or mixed within thereaction chamber by any suitable mechanism. In some embodiments, thetransduction particles can be included in the reagent container 4780and/or the reagent container 4790 within the housing 4741 (or capassembly) as described herein. In such embodiments, the transductionparticles can be added to and/or mixed within the reaction chamber(e.g., the chamber 4732) by the application of a force to an actuator(e.g., the actuator 4750 and/or the actuator 4760), which thereby causesthe transduction particles to be conveyed from the reagent containerinto the reaction container as described herein. In some embodiments,the transduction particles can be conveyed such that the dead volumewithin the cap assembly is between about 30 μL and about 50 μL. In someembodiments, the transduction particles can be conveyed such that the“dead volume” about 40 μL±9 μL. In some embodiments, the transductionparticles can be conveyed such that the dispensed volume is about 285 μLwith a coefficient of variation of about three percent. By limiting thedead volume and/or the part-to-part variation in the dead volume, theaccuracy of delivery, and thus, the accuracy of the assay, can beimproved.

In some embodiments, the transduction particles can be conveyed into thereaction chamber in a manner that reduces the turbulence generatedtherein. For example, in some embodiments, the transduction particlescan be conveyed such that they impinge and/or contact the sidewall ofthe reaction chamber as described herein. In other embodiments, thetransduction particles can be conveyed at a velocity and/or flow rate topromote mixing and/or reduce turbulence. For example, in someembodiments, the mixing of the transduction particles includes conveyingthe transduction particles into the reaction by moving the actuator(e.g., the actuator 4750) linearly at a rate of between about 63 mm persecond and about 81 mm per second In some embodiments, the mixing of thetransduction particles includes conveying the transduction particlesinto the reaction chamber by moving the actuator (e.g., the actuator4750) linearly at a rate of about 72 mm per second.

A first signal associated with the reagent is received, at 115. In someembodiments, the first signal can be an optical signal associated with acolorant included within the reagent. In some embodiments, the firstsignal can be associated with a volume of the assay media within thereaction chamber. In this manner, the first signal can indicate thepresence of the reagent. When the first signal indicates the presence ofthe reagent, the sample and the one or more transduction particles ismaintained to express the one or more reporter molecules when the cellphenotype is present in the same, at 120.

A second signal associated with a quantity of the one or more reportermolecules is received, at 125. The second signal is the detectablesignal, and can be any suitable signal that is produced by certainreporter molecules, such as for example, an optical signal produced by aflash luminescence reaction. In some embodiments, the second signal isassociated with the quantity of reporter molecules within the sample. Insome embodiments, the magnitude of the second signal is independent fromthe quantity of the transduction particles above a predeterminedquantity. Similarly stated, in some embodiments, the strength of thesecond signal is substantially independent from the quantity of thetransduction particles.

Moreover, in some embodiments, the portion of the cell phenotype caninclude a bacteria phenotype that is resistant to an antibioticindividually or in combination with another antibiotic. An antibioticcan include one or more of Beta-lactams, extended-spectrum beta-lactams,Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, any generation ofCephalosporins, Glycopeptides, Lincosamides, Lipopeptide, Macrolides,Monobactams, Nitrofurans, Oxazolidonones, Penicillins, Polypeptides,Quinolones, Fluoroquinolones, Sulfonamides, Tetracyclines, mycobacterialantibiotics, Chloramphenicol, or Mupirocin. In some embodiments, theportion of the cell phenotype can include a bacteria phenotype that areresistant to one or more of cefoxitin, vancomycin, teicoplainin,ampicillin/sulbactam, ciprofloxacin, meropenem, ceftazidime,ceftriaxone, piperacillin/tazobactam, or gentamicin.

In some embodiments, the method 100 can include disposing a substanceinto the sample. The substance is formulated to react with the one ormore reporter molecules to enhance the second signal. For example, insome embodiments, the reporter molecule can be luciferase and the method100 can employ the cap assembly and/or housing 4741 described above. Insuch embodiments, the reagent container 4780 and/or the reagentcontainer 4790 can contain an aldehyde reagent formulated to trigger,initiate and/or catalyze a luminescence reaction that can be detected bythe production of the signal. In some embodiments, the reagent caninclude a 6-carbon aldehyde (hexanal), a 13-carbon aldehyde (tridecanal)and/or a 14-carbon aldehyde (tetradecanal), inclusive of all the varyingcarbon chain length aldehydes therebetween. In some embodiments, theassembly 4700 can be configured to maintain the additional reagent influidic isolation from the sample before being disposed into the sample.In this manner, the timing of the delivery of the additional reagentinto the sample can be controlled. In some embodiments, the system caninclude a mechanism (e.g., mechanism for applying a force to theactuator 4750 and/or the actuator 4760 for adding the additional reagentat any suitable time and/or in any suitable manner to induce thedetectable signal. For example, as described in more detail herein, insome embodiments, the system and/or the testing container can include amechanism for conveying an additional reagent into the sample at apredetermined velocity (or flow rate) to promote the desired level ofmixing.

For example, in some embodiments, the reagent and/or substrate can beconveyed such that it impinges and/or contacts the sidewall of thereaction chamber as described herein. In other embodiments, the reagentand/or substrate can be conveyed at a velocity and/or flow rate topromote mixing and/or reduce turbulence. A step in the luciferasereaction includes the first formation of a complex between luciferaseand flavin mononucleotide. In the absence of a suitable aldehyde (i.e.the substrate), this complex is unable to proceed in the luminescencereaction. The luciferase reaction proceeds and emits light upon theaddition of the aldehyde, and ideally, it is preferable that allcomplexed luciferases be triggered to emit photons simultaneously. Thiswould result in a large flux of photons being emitted in a short periodof time—i.e., a flash of light that can be readily detected. Assupported by the test results presented herein, however, if the reagentand/or substrate is conveyed into the reaction chamber at a rate that istoo high, the amount of light detected will decrease and/or the amountof light detected from replicates will exhibit increased variabilityresulting in an increase in the coefficient of variation associated withlight detection. This reduction in performance is thought to be relatedto splashing and/or formation of bubbles in the solution that can resultwhen the reagent and/or substrate is conveyed at a high velocity.Accordingly, the mixing of the reagent and/or substrate can becontrolled to produce the desired light output performance. For example,in some embodiments, the mixing of the reagent and/or substrate includesconveying the reagent and/or substrate into the reaction chamber bymoving the actuator (e.g., the actuator 4760) linearly at a rate ofbetween about 63 mm per second and about 81 mm per second. In someembodiments, the mixing of the reagent and/or substrate includesconveying the reagent and/or substrate into the reaction chamber bymoving the actuator (e.g., the actuator 4760) linearly at a rate ofabout 72 mm per second.

In some embodiments, a MRSA reporter assay can be developed and/orperformed using any suitable system and method as described herein. Insuch embodiments, a non-replicative transduction particle is developedfrom a S. aureus-specific bacteriophage and the bacterial luciferasegenes luxAB under the control of a constitutive promoter areincorporated. When this transduction particle introduces the reportersystem into S. aureus, the constitutive promoter can express luxABsuitable for reporting on the presence of a viable S. aureus. If inaddition, the antibiotic cefoxitin, or a similar anti-biotic, is alsoadded prior to or simultaneously with mixing the transduction particleswith S. aureus cells, if the cells do not contain and express the mecAgene, no luxAB will be expressed in the assay, thus indicating thatthere is no MRSA. If, however, the cells do contain and express the mecAgene, luxAB will be expressed in the assay, thus indicating that thecells are MRSA (i.e., resistant to inhibition by cefoxitin).

Although container assembly 4700 is shown as including a threadedcoupling between the housing 4741 and the reaction chamber 4732, inother embodiments, a housing can be coupled to a reaction chamber via apress fit. For example, FIG. 24 shows a side, partial cross-sectionalview of a container assembly 5700 according to an embodiment. Thecontainer assembly 5700 can be used with and manipulated by any of theinstruments and/or any of the components described herein and in U.S.patent application Ser. No. 13/802,461, entitled “Systems and Methodsfor Detection of Cells using Engineered Transduction Particles,” whichis incorporated herein by reference in its entirety. In this manner, thecontainer assembly 5700 and any of the container assemblies describedherein can be used to detect and/or identify target cells (e.g.,bacteria) within a sample according to any of the methods describedherein or in the '461 application. For example, in some embodiments, thecontainer assembly 5700 can be used to dispose and/or mix a reagent intoa sample while maintaining fluidic isolation between the container andan outside region. In this manner, the method of cell identification canbe performed in a closed system and/or a homogeneous assay. Similarlystated, in some embodiments the container assembly 5700 is used inmethods of cell identification and/or detection that do not involveremoval of contents from the container assembly 5700, separation of thecontents within the container assembly 5700, washing of the contentswithin the container assembly 5700 and/or rinsing of the contents withinthe container assembly 5700.

The container assembly 5700 includes a housing 5741, a first actuator(not shown), a second actuator 5760, and a reaction chamber (not shown).The housing 5741 is can be removably coupled to the reaction chamber.For example, as shown in FIG. 24, the housing 5741 can be coupled to aproximal portion of the reaction chamber via a press fit portion 5743.Thus, the housing 5741 (and the components disposed therein) can bestored separately from and/or spaced apart from the reaction chamber5732. In this manner, a user can then dispose a sample into the reactionchamber in accordance with the methods described herein (and in the '461application, which is incorporated herein by reference in its entirety),and can then assemble the housing 5741 (or “cap assembly”) to thereaction chamber (or “tube”) and complete the steps for cellidentification, as described herein.

The housing 5741 defines a first reagent volume (not identified)configured to receive a first reagent container (not shown) and a secondreagent volume 5744 configured to receive a second reagent container5790. The housing 5741 includes a first puncturer 5792, a secondpuncturer 5794, a first delivery pathway 5771, and a second deliverypathway 5773. The first puncturer 5792, the second puncturer 5794, thefirst delivery pathway 5771, and the second delivery pathway 5773 aresimilar to the corresponding structure of the housing 4741 describedabove, and are therefore not described in detail.

The press fit portion 5743 includes a recess or groove within which aportion of the reaction chamber can be securely disposed (i.e., to forma press or interference fit). In some embodiments, the press fit portion5743 can include a seal member (e.g., an o-ring or the like) to define asubstantially fluid-tight seal when the housing 5741 is coupled to thereaction chamber.

The second actuator 5760, as shown, is substantially solid, and has awidth substantially similar to a width of the second reagent volume5744. In this manner, undesirable “dead space” within the second reagentvolume 5744 (and/or the first reagent volume, not identified) can belimited. In use, the container assembly 5700 can be actuated in a mannersimilar to that described above with respect to the housing 4741 and/orcap assembly. In particular, the second actuator 5760 can be manipulatedwithin the second reagent volume 5760 to convey a reagent from thesecond reagent volume 5760 to the reaction chamber.

Although the reagent containers (e.g., reagent container 4780, reagentcontainer 4790, reagent container 5780, reagent container 5790) havebeen described and illustrated in positions lateral to each other whendisposed within a housing (e.g., housing 4741), in other embodiments,reagent containers can be disposed within a housing in any suitablemanner or configuration, such as for example, in a verticalconfiguration. For example, FIGS. 25A-C, and FIGS. 26 and 27 show ahousing 6741 (and “cap assembly”) according to an embodiment. Inparticular, FIGS. 25A-C show the housing 6741 in a cross-sectional sideview (FIG. 25A), a cross-sectional front view (FIG. 25B), and a bottomview (FIG. 25C). FIGS. 26 and 27 show the housing 6741 (without thereagent containers) in a cross-sectional front view (FIG. 26) and afront view (FIG. 27).

As shown, the housing 6741 defines a reagent volume 6742 (FIG. 25B)configured to receive a first reagent container 6780 and a secondreagent container 6790. As shown in FIG. 26, the housing 6741 includesfirst rupture member 6798 and a second rupture member 6799. The firstrupture member 6798 and the second rupture member 6799 include a firstpuncturer 6792 and a second puncturer 6794, respectively. The rupturemember 6798 and the rupture member 6799 each define, at least in part, adelivery pathway 6771. The delivery pathway 6771 places the reagentvolume 6742 in fluidic communication with a reaction chamber (notshown). In addition, as shown, the delivery pathway 6771 places thefirst rupture member 6798 and the second rupture member 6799 in fluidiccommunication with each other. In this manner, contents of the reagentcontainer 6780 can communicate (e.g., mix) with contents of the reagentcontainer 6790 before reaching the reaction chamber (not shown) or aportion thereof. Such an arrangement, in some embodiments, can promotemixing and/or minimize aeration, overspray and/or undesirable turbulenceof the contents from the reagent container 6780 and/or the reagentcontainer 6790.

Although shown to be in fluidic communication, in other embodiments, thefirst rupture member 6798 and the second rupture member 6799 can bemaintained in fluidic isolation from each other. For example, in someembodiments, the first rupture member 6798 can define in part a firstdelivery pathway (not shown), and the second rupture member 6799 candefine in part a second delivery pathway (not shown) such that thesecond delivery pathway that is distinct from and/or fluidic isolatedfrom the first delivery pathway.

The rupturing of reagent container 6780 and reagent container 6790 canbe initiated and/or caused at least in part by any suitable means. Forexample, in some embodiments, the housing 6741 can be manipulated suchthat a pressure within an interior portion of the housing 6741 isaltered, resulting in the reagent container 6780 and/or the reagentcontainer 6790 being urged against the first rupture member 6798 and thesecond rupture member 6799, respectively. In this manner, the puncturer6792 of the first rupture member 6798 and/or the puncturer of the secondrupture member 6799 can rupture the first reagent container 6780 and thesecond reagent container 6790, respectively. For example, in someembodiments the housing 6741 can include one or more actuators (similarto the actuator 4750).

While various embodiments have been described above, it should beunderstood that they have been presented by way of example only, and notlimitation. Where methods and/or schematics described above indicatecertain events and/or flow patterns occurring in certain order, theordering of certain events and/or flow patterns may be modified.Additionally certain events may be performed concurrently in parallelprocesses when possible, as well as performed sequentially. While theembodiments have been particularly shown and described, it will beunderstood that various changes in form and details may be made.

Although the puncturers (e.g., puncturer 1792) are described herein asbeing substantially stationary (e.g., fixed) with respect to the housing(e.g., housing 1741), in other embodiments, a puncturer can be moveable(e.g., slideable, rotatable, etc.) with respect to the housing.

In use, any suitable container assembly (e.g., container assembly 1700,2700, 3700, 4700, 5700, etc.) can receive a patient sample (e.g.,bacteria) via any suitable method. For example, in some embodiments, thecontainer assembly can be provided within a kit including additionalcomponents, for example, swabs for collecting patient samples. In suchembodiments, the sample can be delivered to the testing container viathe swab. In other embodiments, the sample can be delivered to thecontainer assembly from a transportation container (e.g., via a pipette,syringe, etc.).

Although the reagent container 4780 and the reagent container 4790 areshown and described has having a specific shape and construction, any ofthe reagent containers (or blister packs) described herein can beconstructed from any suitable material, for example, PVC, and/or from acombination of different materials (e.g., pharmaceutical-gradecopolymer, cyclic olefin copolymer film, Tekniflex COC P12P, PCTFE filmlamination, Tekniflex VA10200). In some embodiments, the reagentcontainers or blister packs described herein can be constructed ofmaterials that are compatible with methods of bioburden reduction thatinclude gamma irradiation.

Moreover, any of the blister packs described herein can be any suitablesize or shape. For example, in some embodiments, a blister pack caninclude a linear portion (e.g., the skirt of the blister pack, a flatsurface) and a non-linear portion (e.g., a rounded surface). In suchembodiments, a blister pack can be configured to limit dead volumetherein (e.g., empty space, a void, a cavity, an area without ofreagent, etc.). In some embodiments, instead of or in addition to thelinear portion, the blister pack can include a concave portion. In thismanner, as the portion is ruptured (e.g., the foil begins to bulge),efficient and/or sufficient contact between the surface of the concaveportion and the puncture member can be established. As such,dispensation of the contents of the blister pack can be maximized and/orreduced dead volume can be achieved.

Any of the reagent containers and/or blister packs described herein(e.g., the reagent container 4780 and/or the reagent container 4790) cancontain a pre-determined amount of any suitable reagent (e.g., assayreagent, antibiotic reagent, transduction particles, substrate reagent,etc.). The pre-determined amount can be measured in any suitable manner,for example, by volume of specific concentrations. Moreover, any of thereagent containers and/or blister packs can include any of thetransduction particles described herein and in U.S. ProvisionalApplication Nos. 61/983,765, entitled “Reagent Cartridge for Detectionof Cells,” filed Apr. 24, 2014; 61/779,177, entitled “Non-ReplicativeTransduction Particles and Transduction Particle-Based ReporterSystems,” filed Mar. 13, 2013; 61/939,126, entitled “Systems and Methodsfor Packaging Nucleic Acid Molecules into Non-Replicative TransductionParticles and Their Use as Cellular Reporters,” filed Feb. 12, 2014; and61/897,040, entitled “Transcript Detection Systems and Methods,” filedOct. 29, 2013, and International Patent Application No.PCT/US2014/026536, entitled “Non-Replicative Transduction Particles andTransduction Particle-Based Reporter Systems,” filed Mar. 13, 2014, eachof which is incorporated herein by reference in its entirety

Although the container assemblies, systems and methods are describedherein as being used to detect and/or identify target cells usingnon-replicative transduction particles, in other embodiments, any of thecontainer assemblies and systems described herein can be used inconjunction with any suitable reagents to detect a target bacteria. Forexample, in some embodiments, the assemblies and systems describedherein can be used in conjunction with replication-competenttransduction particles, such as, for example, a traditional phagereporter.

For example, in some embodiments, a housing or “cap assembly” (e.g., thehousing 4741) contains two reagent volumes (e.g., the volumes 4742 and4744) and/or two reagent containers (e.g., the reagent containers 4780and 4790). The first reagent volume and/or reagent container contains anengineered luciferase-reporter bacteriophage, such as for example theNanoluc® luciferase produced by Promega Corp. The second reagent volumeand/or second reagent container contains a substrate, such asfurimazine. In some embodiments, the substrate can be formulated forspecific compatibility with the luciferase-reporter bacteriophage (e.g.,the Nanoluc®) contained within the first reagent volume.

In use, after the sample is added to the reaction chamber and the capassembly is coupled thereto, the method includes adding the contents ofthe first reagent volume to the reaction chamber. The sample and thefirst reagent (e.g., the reporter phage, such as the Nanoluc® reporterphage) are maintained at or above a predetermined temperature for apredetermined time period (i.e., the sample is incubated). If the samplecontains bacteria that the reporter phage is designed to target, thenthe reporter phage causes viable target bacteria to express theluciferase during the incubation period. After the incubation period,the contents of the second reagent volume are added to the reactionchamber providing the substrate (i.e., furimazine) that can react withany expressed luciferase and generate a luminescent signal therebyindicting the presence of the target bacteria in the sample. The lightoutput produced during this assay (and any of the assays describedherein) can be detected using any suitable instrument, such as theinstruments described in the '461 application.

In some embodiments, a method includes using a luciferase reporter phageto identify the presence of antibiotic resistant bacteria. For example,in some embodiments, a reaction chamber (e.g., the reaction chamber4732) can include an antibiotic, for example, as a dry reagent. Anyantibiotics of the type described herein can be used. A housing or “capassembly” (e.g., the housing 4741) is configured to be removably coupledto the reaction chamber and contains two reagent volumes (e.g., thevolumes 4742 and 4744) and/or two reagent containers (e.g., the reagentcontainers 4780 and 4790). The first reagent volume and/or reagentcontainer contains any suitable reporter phage. The second reagentvolume and/or second reagent container contains a substrate, such asluciferin.

In use, after the sample is added to the reaction chamber and the capassembly is coupled thereto, the method includes adding the contents ofthe first reagent volume to the reaction chamber. The sample and thefirst reagent (e.g., the reporter phage) are maintained at or above apredetermined temperature for a predetermined time period (i.e., thesample is incubated). In this manner, any bacteria present within thesample that are resistant to the antibiotic are able to propagate andexpress luciferase. Conversely, those bacteria within the sample thatare sensitive to the antibiotic do not propagate and/or do not expressluciferase and/or are otherwise unable to successfully mediate aluminescence reaction. After the incubation period, the contents of thesecond reagent volume are added to the reaction chamber providing thesubstrate that can react with any expressed luciferase to generate aluminescent signal thereby indicting the presence of the target bacteriain the sample. The light output produced during this assay (and any ofthe assays described herein) can be detected using any suitableinstrument, such as the instruments described in the '461 application.

In other embodiments, a luciferase reporter assay can employ thebacterial luciferase genes luxAB as the first reagent, and itssubstrate, an aldehyde such as decanal, as the second reagent. In yetother embodiments, a luciferase reporter assay can employ the bacterialluciferase genes operon including the genes luxCDEAB, thus eliminatingthe need for the addition of a substrate (e.g., aldehyde) since theoperon contains genes that enable the target bacteria to produce thealdehyde. In such embodiments, the cap assembly need only define onereagent volume or include one reagent container. Such methods can beused in conjunction with an antibiotic and/or antimicrobial compound, asdescribed herein.

Although the container assemblies, systems and methods are describedherein as being used to detect and/or identify target cells, such asbacteria, in other embodiments, any of the container assemblies, systemsand methods described herein can be used in conjunction with anysuitable homogenous assay. Moreover, although the container assemblies,systems and methods are described herein as being used to detect and/oridentify target cells, such as bacteria, in other embodiments, any ofthe container assemblies, systems and methods described herein can beused in conjunction with any assay that incorporates a “switchablesignal”—i.e., reporter system that enables a homogeneous assay where aspecific signal can be generated and detected without the need forwashing or separation steps. Similarly stated, any of the containerassemblies, systems and methods described herein can be used inconjunction with any suitable assay in which a signal is not producedunless or until a reaction with an analyte occurs and/or in which noamount of a reporter is present in the sample until the conditions aresuch that the reporter is produced. Moreover, although the “switchable”reporter molecules are described herein as being molecules that can beexpressed from reporter genes encoding enzymes mediating luminescencereactions, in other embodiments a switchable reporter can be mediated bythe direct addition of a molecule that is ‘switched on’ to produce asignal upon, for example, a conformational change mediated by thebinding to a target analyte, such as for example, the switchable aptamerdesigned to detect S-adenosylmethionine consisting of an RNA termedSpinach and the fluorophore 3,5-difluoro-4-hydroxybenzylideneimidazolinone (DFHBI) designed to include a transducer element thatbinds to S-adenosylmethionine that causes as a conformational changethat allows for fluorophore activation as described in “Fluorescenceimaging of cellular metabolites with RNA, Science. March 2012, vol. 335,no. 6073, 9, pp. 1194.” In yet other embodiments, a switchable reporterinclude anything that exhibits a first signal before reacting with ananalyte and a second (different) signal after reacting with the analyte.

In some embodiments, the container assemblies and systems describedherein can be used in a hygiene assay to determine the presence ofliving (or previously living) organisms by detecting the presence ofadenosine triphosphate (ATP) within the sample. In such embodiments, asample of unknown hygiene is added to a reaction chamber (e.g., thereaction chamber 4732). The method further includes attaching a housingor “cap assembly” (e.g., the housing 4741) to the reaction chamber. Asdescribed above, the housing contains two reagent volumes (e.g., thevolumes 4742 and 4744) and/or two reagent containers (e.g., the reagentcontainers 4780 and 4790). The first reagent volume and/or reagentcontainer contains a nutrient media formulated such that any organismspresent in the sample will remain metabolically active. The nutrientmedia can be similar to and/or contain any of the nutrients orcompositions of the transport media described herein. The second reagentvolume and/or second reagent container contains a formulation includinga eukaryotic luciferase enzyme, luciferin, and a lysis reagent. Asdescribed below, when ATP is present a light output is produced (i.e.,via a luminescence reaction) that can be detected using any suitableinstrument, such as the instruments described in the '461 application.

In use, after the sample is added to the reaction chamber and the capassembly is coupled thereto, the method includes adding the contents ofthe first reagent volume to the reaction chamber to provide nutrientsfor any organisms in the sample to remain metabolically active. In someembodiments, the sample can be incubated for a time period to allow anyorganisms in the sample to grow. Such incubation can be performed usingany of the instruments described in the '461 application for anysuitable time and at any suitable temperature. The contents of secondreagent volume can then be added to the reaction chamber. The lysisreagent (included within the second reagent chamber) is formulated torelease adenosine triphosphate produced by viable organisms that mayexist within the sample. If the sample contains any viable organismsthen the luciferase and luciferin will react along with extractedadenosine triphosphate causing a luminescent reaction and thusindicating the presence of viable organisms in the reaction.

In other embodiments, a method of performing a hygiene assay need notinclude adding a nutrient medium, as indicated above. Thus, in someembodiments, the cap assembly can include only a single reagent volume,actuator and/or reagent container. In such embodiments, the method caninclude reporting on the presence of viable organisms directly from asample without an incubation step.

In other embodiments, a method of performing a hygiene assay can includedelivering a variety of different compositions using a cap assembly ofthe types described herein. In this manner, certain compositions used inthe assay can be stored separately from and/or spaced apart from othercompositions. For example, such methods that employ the cap assembliesdescribed herein can facilitate separate storage of the lysis (orextraction) reagent, which can limit the likelihood that the lysis agentwill negatively impact the performance of the luciferase enzyme and/orthe luciferin. For example, in some embodiments, the luciferin and/orthe luciferase can be incorporated into a dry reagent and placed intothe reaction chamber. This arrangement also accommodates the use ofluciferin and/or luciferase formulations that are not stable in liquidform. For example, in some embodiments, the luciferin can be included asa dried reagent within the reaction chamber, the first reagent volume(of the cap assembly) can include the lysis reagent, and the secondreagent volume (of the cap assembly) can include the eukaryoticluciferase enzyme. In use, the lysis reagent can be added at a differenttime than the luciferase enzyme, thus allowing for a controlled lysisperiod prior to adding the luciferase enzyme. In other embodiments, theluciferase enzyme can be included as a dried reagent within the reactionchamber, the first reagent volume (of the cap assembly) can include thelysis reagent, and the second reagent volume (of the cap assembly) caninclude the luciferin. In yet other embodiments, both the luciferaseenzyme and the luciferin can be included as a dried reagent within thereaction chamber and a reagent volume (of the cap assembly) can includethe lysis reagent.

In some embodiments, a method of performing a hygiene assay can includeexposing the sample to an antimicrobial compound. The antimicrobialcompound can include any substance, such as for example, an antibiotic,formulated and/or selected to kill non-targeted organisms (e.g.,bacterial strains or the like). In this manner, the method can be usedto indicate the presence of viable organisms in the reaction that areinsensitive or resistant to the antimicrobial compound. In suchembodiments, the antimicrobial compound can be included within thereaction chamber, for example, in a dried form. Thus, in someembodiments, a method includes adding a sample of unknown hygiene to thereaction chamber containing the antimicrobial compound. A housing or“cap assembly” (e.g., the housing 4741) is attached to the reactionchamber. As described above, the housing contains two reagent volumes(e.g., the volumes 4742 and 4744) and/or two reagent containers (e.g.,the reagent containers 4780 and 4790). The first reagent volume and/orreagent container contains a nutrient media formulated such that anyorganisms present in the sample will remain metabolically active. Thenutrient media can be similar to and/or contain any of the nutrients orcompositions of the transport media described herein. The second reagentvolume and/or reagent container contains a formulation including aeukaryotic luciferase enzyme, luciferin, and a lysis reagent. Asdescribed below, when ATP is present a light output is produced (i.e.,via a luminescence reaction) that can be detected using any suitableinstrument, such as the instruments described in the '461 application.

After the sample is added to the reaction chamber and the cap assemblyis coupled thereto, the contents of the first reagent volume can beadded to the reaction chamber to provide nutrients for any organisms inthe sample (that are resistant to the antimicrobial compound) to remainmetabolically active. In some embodiments, the sample can be incubatedfor a time to allow any organisms that are resistant to theantimicrobial compound in the sample to grow. Such incubation can beperformed using any of the instruments described in the '461 applicationfor any suitable time and at any suitable temperature. The contents ofsecond reagent volume can then be added to the reaction chamber. Thelysis reagent (included within the second reagent chamber) is formulatedto release adenosine triphosphate produced by viable organisms that mayexist within the sample (i.e., those that are insensitive or resistantto the antimicrobial compound). If the sample contains any viableorganisms then the luciferase and luciferin will react along withextracted adenosine triphosphate causing a luminescent reaction and thusindicating the presence of such viable organisms in the reaction.

In some embodiments, the container assemblies and systems describedherein can be used to detect the presence of certain enzymes in asample. In this manner, the function and/or characteristics of anyorganisms present within a sample can be determined. For example, insome embodiments, a method includes determining the presence of abetalactamase enzyme, which can be indicative of a bacteria that isresistant to certain antibiotics. In such embodiments, a reactionchamber (e.g., the reaction chamber 4732) can include a caged luciferasesubstrate (e.g., in a dried form), such as for example, a cagedD-luciferin molecule such as those β-lactam-d-luciferin (Bluco)described in “Hequan Yao et al., A Bioluminogenic Substrate for In VivoImaging of Beta-Lactamase Activity, Angewandte Chemie InternationalEdition, August 2007, vol. 46, pp. 7031-7034”. The caged luciferasesubstrate can be designed and/or engineered to have limited or noreactivity as the luciferase substrate unless a betalactamase enzymefirst reacts with the caged luciferase substrate such that it un-cagesthe substrate. A housing or “cap assembly” (e.g., the housing 4741) isconfigured to be removably coupled to the reaction chamber. The housingcontains two reagent volumes (e.g., the volumes 4742 and 4744) and/ortwo reagent containers (e.g., the reagent containers 4780 and 4790). Thefirst reagent volume and/or reagent container contains a cell lysisreagent. The second reagent volume and/or second reagent containercontains a bioluminescent molecule such as Renilla luciferase.

After the sample is added to the reaction chamber and the cap assemblyis coupled thereto, the method includes adding the contents of the firstreagent volume to the reaction chamber. In this manner, any cells thatmay be present in the sample are lysed. If the sample contains targetcells that express the betalactamase, such lysing of the cells releasesthe betalactamase and other intracellular molecules. The method thenincludes adding the contents of the second reagent volume to thereaction chamber. If betalactamase is present, it is able to un-cage thecaged luciferin and allow the un-caged luciferase substrate to reactwith the luciferase (added from the second reagent volume, e.g., theRenilla luciferase) thereby producing a luminescence signal that isindicative of the presence of the betalactamase enzyme in the sample.The light output produced during this assay (and any of the assaysdescribed herein) can be detected using any suitable instrument, such asthe instruments described in the '461 application.

In other embodiments, the caged luciferase substrate can be included inany suitable portion (or volume) of the container assembly and/or can beadded at any suitable juncture of the method. For example, in someembodiments, a first reagent volume of a cap assembly can include anon-replicative transduction particle designed to express Renillaluciferase in the target cell. A second reagent volume can include acaged luciferase substrate designed to have limited or no reactivity asthe luciferase substrate unless a betalactamase enzyme first reacts withthe caged luciferase substrate such that it un-cages the substrate.

After the sample is added to the reaction chamber and the cap assemblyis coupled thereto, the method includes adding the contents of the firstreagent volume to the reaction chamber. The resulting solution is thenmaintained at or above a predetermined temperature for a time period(i.e., the solution is incubated). In this manner, if the samplecontains target cells, the non-replicative transduction particle is ableto cause the target cell to express Renilla luciferase. The method thenincludes adding the contents of the second reagent volume to thereaction chamber. If the target bacteria produces a betalactamase, thebetalactamase is able to un-cage the caged luciferase substrate andallow the un-caged luciferase substrate to react with the luciferasethereby producing a luminescence signal that is indicative of thepresence of the betalactamase enzyme in the sample. The light outputproduced during this assay (and any of the assays described herein) canbe detected using any suitable instrument, such as the instrumentsdescribed in the '461 application

Although the methods are shown and described as determining the presenceof a betalactamase enzyme, in other embodiments, methods and system canbe used to determine the presence of any suitable enzyme. For example,in some embodiments, a method includes determining the presence of acarbapenemase enzyme. In such embodiments, a reaction chamber (e.g., thereaction chamber 4732) can include a dried carbapenemase substrate suchas a carbapenem or cephamycin. A housing or “cap assembly” (e.g., thehousing 4741) is configured to be removably coupled to the reactionchamber. The housing contains two reagent volumes (e.g., the volumes4742 and 4744) and/or two reagent containers (e.g., the reagentcontainers 4780 and 4790). The first reagent volume and/or reagentcontainer contains a cell lysis reagent. The second reagent volumeand/or second reagent container contains a reagent containing pHindicator formulated such that when added to the sample, the reactionwill change color when the pH of the reaction mixture is comprisedbetween 6.4 and 8.4.

After the sample is added to the reaction chamber and the cap assemblyis coupled thereto, the method includes adding the contents of the firstreagent volume to the reaction chamber. In this manner, any cells thatmay be present in the sample are lysed. If the sample contains targetcells that express the carbapenemase, such lysing of the cells releasesthe carbapenemase and other intracellular molecules. The method thenincludes adding the contents of the second reagent volume to thereaction chamber. If carbapenemase is present, it reacts with thecarbapenemase substrate and causes a color change via the pH indicatorwherein a color change indicates the presence of carbapenemase-producingbacteria in the sample. The color change produced during this assay (andany of the assays described herein) can be detected using any suitableinstrument, such as the instruments described in the '461 application.

In some embodiments, the container assemblies and systems describedherein can be used in a DNA sequencing assay. In such embodiments, areaction chamber (e.g., the reaction chamber 4732) contains acomposition of dried aptamer molecules and a dried fluorophore. Thedried aptamer molecules are formulated, engineered and/or designed tobind to a target sequence of DNA. The dried fluorophore (also referredto as a dye) is designed to preferentially fluoresce when bound to acomplex formed by the binding of the aptamer to the target DNA sequence.A housing or “cap assembly” (e.g., the housing 4741) is removablycoupleable to the reaction chamber. As described above, the housingcontains two reagent volumes (e.g., the volumes 4742 and 4744) and/ortwo reagent containers (e.g., the reagent containers 4780 and 4790). Thefirst reagent volume and/or reagent container contains a buffer solutiondesigned to produce and/or promote conditions that are favorable foraptamer/target DNA/fluorophore binding. The second reagent volume and/orsecond reagent container contains a formulation containing anoligonucleotide designed to bind to the target DNA molecule and displace(e.g., “out-compete”) an aptamer that may already be bound to the targetDNA molecule.

In use, after the sample is added to the reaction chamber and the capassembly is coupled thereto, the method includes adding the contents ofthe first reagent volume to the reaction chamber. Because the additionof the first reagent produces conditions favorable for aptamer/targetDNA/fluorophore binding, if the sample contains target DNA, then bindingwill occur and a fluorescence signal from the complexed fluorophore canbe detected. In this manner, the aptamer molecules can be considered asa “switchable” aptamer. After time period has elapsed, the contents ofthe second reagent volume can be added to the reaction chamber. If thefluorescence signal is eliminated after the addition of theoligonucleotides introduced from the second reagent volume, then if theloss of signal is due to the displacement of the aptamer from the targetDNA (and therefore displacement of the fluorophore from the nowun-complexed aptamer) by the oligonucleotide. Such loss of signal servesas confirmation that the initial fluorescent signal was specifically dueto the complexing of the aptamer to the target DNA. The light outputproduced during this assay (and any of the assays described herein) canbe detected using any suitable instrument, such as the instrumentsdescribed in the '461 application.

In some embodiments, DNA sequence detection systems and methods caninclude detection of DNA within live cells. For example, in someembodiments, the method described above can be modified such that thereaction chamber (e.g., the reaction chamber 4732) contains a driedfluorophore that is formulated and/or engineered such that it can enterlive cells when a sample containing live cells is added to a reactionchamber. Moreover, the fluorophore is also designed to preferentiallyfluoresce when bound to a complex formed by the binding of the aptamerto the target DNA sequence. The housing or “cap assembly” (e.g., thehousing 4741) used in conjunction with the method contains two reagentvolumes (e.g., the volumes 4742 and 4744) and/or two reagent containers(e.g., the reagent containers 4780 and 4790). The first reagent volumeand/or reagent container contains a liposome and an aptamer. The aptamercan be similar to those described above, and are formulated, engineeredand/or designed to bind to a target sequence of DNA. The liposomes cancarry the aptamers directly into the live cells, or can carry a DNAsequence designed to express the aptamers within the live cells. Thesecond reagent volume and/or second reagent container contains a lysisreagent to release the molecules within the live cell and anoligonucleotide designed to displace the aptamer from the target DNAsequence (similar to that described above).

In use, after the sample is added to the reaction chamber and the capassembly is coupled thereto, the contents of the first reagent volumecan be added to the reaction chamber. After delivery of the aptamer intothe reaction chamber, the aptamer is delivered into the cells via theliposome contained in the first reagent volume. In other embodiments,any other suitable mechanism for transporting the aptamers into thecells can be used. Moreover, in some embodiments, the liposomes cancarry and/or transport the aptamers directly into the living cells,whereas in other embodiments, the liposomes can carry and/or transport aDNA sequence designed to express the aptamers within the live cells.After the aptamer is inside of the live cell, it can complex with atarget DNA sequence allowing for the complex to bind the fluorophore andproduce a fluorescent signal that is indicative of the aptamer bindingto the target DNA sequence. After a time period, the contents of thesecond reagent volume can be added to the reaction chamber. The additionof the lysis reagent releases the molecules within the live cell and theoligonucleotide can thus displace the aptamer from the target DNAsequence. Accordingly, if the fluorescence signal is eliminated afterthe addition of the oligonucleotides introduced from the second reagentvolume, then the loss of signal is due to the displacement of theaptamer from the target DNA (and therefore displacement of thefluorophore from the now un-complexed aptamer) by the oligonucleotide.This serves as confirmation that the initial fluorescent signal wasspecifically due to the complexing of the aptamer to the target DNA. Thelight output produced during this assay (and any of the assays describedherein) can be detected using any suitable instrument, such as theinstruments described in the '461 application.

In some embodiments, the container assemblies and systems describedherein can be used in an assay to determine the transcription activityof a sample. In such embodiments, a sample can be disposed within areaction chamber (e.g., the reaction chamber 4732). A housing or “capassembly” (e.g., the housing 4741) is removably coupleable to thereaction chamber. The housing contains two reagent volumes (e.g., thevolumes 4742 and 4744) and/or two reagent containers (e.g., the reagentcontainers 4780 and 4790). The first reagent volume and/or reagentcontainer contains liposomes carrying molecular beacons designed tofluoresce when the beacon has bound to a target RNA transcript sequence.The second reagent volume and/or second reagent container contains aformulation containing a lysis reagent and oligonucleotides designed topreferentially bind to the target RNA sequence and displace a boundmolecular beacon (e.g., “out-compete” the beacon).

After the sample is added to the reaction chamber and the cap assemblyis coupled thereto, the method includes adding the contents of the firstreagent volume to the reaction chamber. The liposomes added can deliverthe molecular beacons into live cells that may be present in the sample.If the sample contains live cells of bacteria that are transcribing thetarget RNA, then the molecular beacons can bind to the target RNAsequence and produce a fluorescent signal. The fluorescent signal can bedetected using any suitable instrument, such as the instrumentsdescribed in the '461 application. The contents of the second reagentvolume are then added to the reaction chamber. The addition of the lysisreagent releases the molecules within the live cell and theoligonucleotide can thus displace the molecular beacon from the targetRNA sequence. Accordingly, if the fluorescence signal is eliminatedafter the addition of the oligonucleotides introduced from the secondreagent volume, then the loss of signal is due to the displacement ofthe molecular beacon from the target RNA (and therefore the re-quenchingof the displaced molecular beacon) by the oligonucleotide. This servesas confirmation that the initial fluorescent signal was due to thecomplexing of the molecular beacon to the target RNA. The light outputand/or change in light output produced during this assay (and any of theassays described herein) can be detected using any suitable instrument,such as the instruments described in the '461 application.

In yet other embodiments, the cap assemblies, containers and methodsdescribed herein need not be used to determine the presence of cells orbiologic activity. For example, in some embodiments, the containerassemblies and systems described herein can be used in a titration assayto determine, for example, the pH of a sample. In such embodiments, asample of unknown pH is added to a reaction chamber (e.g., the reactionchamber 4732) containing a dried pH indicator dye such as bromothymolblue. A housing or “cap assembly” (e.g., the housing 4741) is attachedto the reaction chamber. As described above, the housing contains tworeagent volumes (e.g., the volumes 4742 and 4744) and/or two reagentcontainers (e.g., the reagent containers 4780 and 4790). One of thereagent volumes and/or containers contains a known concentration ofhydrochloric acid, and the other reagent volume and/or containercontains a known concentration of sodium hydroxide.

In use, after adding the sample, the color of the solution in thereaction chamber can be determined (e.g., using any suitable instrument,such as the instruments described in the '461 application, which cancontain a photodetector capable of determining the sample color). If thesample pH is neutral (6<pH<7.6), then the solution within the chamber(i.e., the solution of the sample and the dried reagent within thechamber) is green. If, however, the sample pH is >7.6 then the solutionis blue. When the instrument detects the sample color as blue, thereagent in the first reagent volume (i.e., the known concentration ofhydrochloric acid) can be added to the reaction chamber. The addition ofthe first reagent can be performed at any suitable rate and/or in anysuitable amount. For example, in some embodiments, a predeterminedamount of the first reagent (HCl) can be added. If the reaction turnsfrom blue to yellow, then the sample contains at least an amount ofhydroxyl ions that is equivalent to the concentration of hydrochloricacid in the first reagent volume. Thus, when the instrument detects achange in the color (e.g. from blue to yellow), an output indicating thepH and/or ion concentration can be produced.

If the pH is <6 then the reaction is yellow and the reagent in thesecond reagent volume (i.e., the known concentration of sodiumhydroxide) can be added to the reaction chamber. If the reaction turnsfrom yellow to blue, then the sample may contain at least an amount ofhydrogen ions that is equivalent to the concentration of sodiumhydroxide in the second reagent volume. Thus, when the instrumentdetects a change in the color (e.g. from yellow to blue), an outputindicating the pH and/or ion concentration can be produced.

In yet other embodiments, any of the reagent volumes or reagentcontainers described herein can contain any suitable reagent tofacilitate the use therein for any suitable assay. For example in someembodiments, a reagent volume or reagent container can include a varietyof different dyes or indicators. Such dyes can include, for example,membrane dyes, lipophilic stains (e.g., “Nile red” or9-diethylamino-5-benzo[α]phenoxazinone), a lipophilic cationicindocarbocyanine dye (e.g., “DiI” or(2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole;perchlorate) and/or a cell-permeant dye that can be used to determinecell viability (e.g., Calcein AM, produced by Life Technologies).

Although various embodiments have been described as having particularfeatures and/or combinations of components, other embodiments arepossible having a combination of any features and/or components from anyof embodiments as discussed above. For example, in some embodiments, theactuator 4750 and/or the actuator 4760 of the cap assembly describedabove can include a concave engagement feature, such as the engagementportion 3754 described above with respect to the actuator 3750.

Analysis of the Collection Tool

The method 200 described above includes transferring the contents (e.g.,transport media) disposed within the interior region of a transportcontainer (e.g., the transport chamber 4014) to a reaction chamber(e.g., the reaction chamber 4732). Transferring the transport mediaincludes transferring the patient sample (e.g., collected using acollection tool, such as a swab) from the transport chamber to thereaction chamber. In some embodiments, transferring the contents caninclude communicating the target bacteria released from the collectiontool into the transport media, via a transfer tool (e.g., a pipette), tothe reaction chamber. Thus, in some embodiments, methods can employ acollection tool that is effective for both (1) collecting a sample(e.g., from a nasal cavity of a patient) and (2) releasing the collectedsample into the transport chamber, container assembly and/or reactionchamber. In particular, methods can employ a collection tool that issuitable for such methods in which the sample can include very lowlevels of the target bacteria (i.e., “low loads”) and/or that employlimited incubation time. In this manner, method of detecting targetcells can be effective even when the amount of target cells availablefor detection is limited.

For example, collection tools having a collection portion constructedfrom a wound material, such as wound Rayon or Dacron, may provide forpatient comfort and/or efficient collection of the sample, but may notrelease a sufficient amount of the collected sample into the transportmedium and/or transport chamber to be effective in the methods describedherein. See, e.g., “Comparison of Rayon and Dacron Swabs in Amies Mediumfor Bordetella pertussis Transport,” J. Stephen Thompson et al., ASM99th General Meeting, May 1999; “Why Flocked Swabs are Superior to FiberWrapped Swabs and Foam Swabs and How They Can Improve Infectious DiseaseDiagnosis,” Copan Innovation, Brescia, Italy, retrieved online at[http://www.mls.be/nieuwsbrieven/css/Why-Flocked-Swabs-are-Superior-to-Fiber-and-Foam.pdf](“Copan Innovation”). Moreover, collection tools having a collectionportion constructed from a foam material are consider has having poorabsorption properties, and are thus often not used in bacterial assays.See Copan Innovation. Accordingly, tests were conducted to evaluate thecollection tool (and more specifically the construction of thecollection portion or “swab”) to determine an appropriate collectiontool for the assays and methods described herein.

A first test involved a comparison of cell recovery during transfer ofcells using Rayon wound swabs (Becton Dickinson swab BD-220115), Nylonfoam swabs (Puritan swab 25-1506 1PF) and a direct transfer of cells. Aschematic illustration of the test procedure and the test results areshown in FIG. 28. As shown, the amount of cells released into thesolution using foam swabs is approximately seven times greater than theamount of cells released using the wound swabs. In fact, the release ortransfer efficiency of the foam swabs was comparable to that from adirect transfer of cells (e.g., via pipetting) into the solution.Accordingly, although wound swabs may provide for better liquidabsorption (e.g., for use in collecting a sample), the release ortransfer performance of wound swabs was inferior to that of foam swabs.

A second test involved comparing the signal output (i.e., Relative LightUnits, or RLU) associated with a solution containing target cellstransferred via a wound swab, a “flocked” swab and a foam swab. In thismanner, by comparing the signal output, the second test was closelyassociated with the methods of detection described herein. As shown inFIG. 29, the second test included placing a swab into a known samplecontaining an amount of target cells. Each swab was then placed into acontainer containing amount of a transport media. In this manner, thecontainer functions in a manner similar to any of the transport chambersdescribed herein. In addition to the three different swabs, the testalso included a “control” test, in which a portion of the sample wasdirectly transferred into the container.

Each container was maintained in controlled conditions for apredetermined time or “incubation” period. The test included anincubation period of two hours and an incubation period of 20 hours.After completion of the incubation period, a controlled amount of thetransport media was transferred to an assay plate for a manual assay. Areagent (i.e., a substrate) was added to the assay plate to react withthe plurality of reporter molecules to enhance the luminescence signal.The luminescence signal was then recorded.

FIGS. 30A and 30B are graphs of the amount of light output (for both thetwo and 20 hour incubation times) and the amount of target cell recovery(in a percentage “colony forming units” resulting from the second test.FIG. 31A is a table showing the light output results for each individualtest run (identified as tests S30, S32 and S60), and FIG. 31B is a graphof the data shown in the table in FIG. 31A. As the results of the secondtest show, for the two hour incubation time, the foam swab resulted inthe production of more light output than did either the wound or flockedswab. Thus, although some assays are performed with wound swabs (forpossible improved sample collection characteristics) or flocked swabs(for possible improved performance in sample collection and/or celltransfer), the surprising result is that the use of a collection toolhaving a collection portion constructed from a foam material can producea greater signal. This advantage is particularly important where methodsinvolve low cell loading and/or low nominal light outputs, which is thecase with many of the methods described herein.

Similarly stated, these results demonstrate that at low sample levels(e.g., after a short time period), the performance of the foam swab issuperior to that of a flocked swab. As such, in some embodiments, themethods described herein can include a collection tool selectedspecifically for use with incubation times that produce a low signal. Insuch embodiments, for example, a foam swab can be selected for a shortincubation time and/or when there is limited amount of sample when it isdetermined that a foam swab performs adequately and/or performs betterthan other swab types at such short incubation times.

Because the second test indicated that under certain conditions the useof either a foam swab or a flocked swab was superior to the use of awound swab, additional tests were conducted assess the performance offlocked swabs in different transport media. In particular, a third testwas run comparing the signal output (i.e., Relative Light Units, or RLU)associated with a solution containing target cells and a “flocked” swabusing two different types of transport media. The first media wasidentified as BSS M64 and the second media was identified as TSB Mod.The constituents of the two media are identified below in Table 1.First, the assay was run on the solution in the absence of any swab, andeach solution produced a sufficient amount of light. Second, the assaywas run with flocked swabs having been disposed within the solution. Inthis instance, the solution using the TSB Mod media failed to producesufficient light output to complete the assay. Finally, the flockedswabs were soaked with the each of the two transport media and were thenused to transfer cells to a conditioned media. In this instance, theswabs that were soaked in the TSB Mod solution failed to producesufficient light output to complete the assay. Thus, although flockedswabs were shown in the second series of tests to be comparable to thefoam swabs, the third series of tests showed that flocked swabs whenused with the TSB Mod transport media did not perform adequately.

TABLE 1 Components per liter of TSB grams Total amount BSS M64 EnzymaticDigest of Casein 17 1.70% Enzymatic Digest of Soybean Meal 3 0.30%Glucose 2.5 0.30% Dipotassium Phosphate 2.5 0.30% Sodium Chloride 50.50% TSB Mod CaCl2 0.55 0.005M MgCl2 0.952 0.01M BGP 12.96 0.06M

Finally, a fourth series of tests was conducted assess the performanceof flocked swabs and foam swabs when collecting a known sample via nasalsampling. The third test was run comparing the signal output (i.e.,Relative Light Units, or RLU) associated with target cells collectedfrom nasal samples. As indicated in Table 2 below, the use of a flockedswab recovered on average about 50,000 more CFU/ml of cells than did theuse of a foam swab.

TABLE 2 Sample # Foam Flocked Flocked − Foam 1 24,000 180,000 156,000 5400 6,400  6,000 6 800 600    (200) 7 10,000 140,000 130,000 8 10,000220,000 210,000 9 2,600 5,800  3,200 10 800 600    (200) 11 800 200   (600) 12 34,000 32,000    (2,000) 13 400 400 — Average  50,220

Analysis of the Rate of Delivery of the Substrate

The methods described above include mixing a substrate with a sample ata predetermined rate. More particularly, in some embodiments, abacterial luciferase reporter transduction particle can be employed.These reporters cause the expression of a bacterial luciferase such asthat from the organism V. fischeri. Bacterial luciferase is comprised ofthe luxA and luxB genes encoding LuxA and LuxB proteins that combine toform the active luciferase enzyme. LuxAB catalyzes a luminescentreaction in the presence of oxygen, reduced flavin mononucleotide(FMNH2, supplied by the host cell), and an aldehyde such as tridecanal(supplied exogenously and which readily penetrates into viable bacterialcells).

Accordingly, during such methods or assays, bacterial luciferase isexpressed and the luciferase molecules complex FMNH2 molecules. Thesecomplexes accumulate and when an aldehyde is added, the luminescencereaction proceeds. Ideally, it is preferable that all complexedluciferases are triggered to emit photons simultaneously. In thismanner, a large flux of photons is emitted in a short period oftime—i.e., a flash of light is produced that can be readily detected,especially when there is a low load of target cells. It is understoodthat if the complexed luciferases emit light in an un-synchronizedmanner, the photons are emitted over an extended period of time therebynot producing a flash.

Because the light emission kinetics is mediated by the availability ofaldehyde (i.e., the substrate), under ideal conditions it is desirableto deliver the aldehyde instantaneously to an entire volume of areaction. Injecting aldehyde into the reaction at a rapid speed canapproach this ideal situation. Therefore, it can be reasoned that fasterinjection speeds will result in more optimal flash reactions. Indeed, astudy that examined the effect of injection speed on light output foundthat increasing injection speed resulted in greater light output whenmeasuring the peak value of light production. However, at a certainpoint, an increase in injection speed was found to result in lower lightoutput and/or greater variability in the results. This phenomenon isattributed to splashing and bubble formation in the reaction that servesto perturb the detection of the light produced. Therefore, a desiredrange of injection speed (expressed as the speed of the actuator) wasfound where maximal light output is attained.

The test results are summarized in FIG. 31, which is a bar chart showingthe average maximum RLU obtained from luciferase expressing cells afterinjecting aldehyde at varying speeds (the speeds are presented in stepsper second, where one step is 0.0254 mm). Note that the RLU values areexpressed as a percentage or the maximum RLU value obtained in thisstudy. As shown, an optimum RLU output was observed at 3,200 steps/secwhere the RLU values were maximum and the variability in light output(expressed as a coefficient of variation) was at a minimum. Furthertesting identified an optimal range of between about 2,500 steps/sec(63.5 mm/sec) and about 3,200 steps/sec (81.3 mm/sec). Thus, in someembodiments, the substrate is mixed by moving the actuator linearly at arate of about 2,850 steps/sec (72.4 mm/sec).

What is claimed is:
 1. A method, comprising: disposing a sample into areaction chamber, the reaction chamber packaged to contain a reagentformulated to mix with the sample to form an assay media; mixing withthe sample in the reaction chamber a plurality of transduction particlesassociated with a cell phenotype, the plurality of transductionparticles engineered to include a nucleic acid molecule formulated tocause the cell phenotype to produce a plurality of reporter moleculescapable of generating a detectable signal, the reagent formulated tosuppress the detectable signal in a portion of the cell phenotype;receiving a first signal associated with the reagent; maintaining, whenthe first signal indicates the presence of the reagent, the sample andthe plurality of transduction particles to express the plurality ofreporter molecules when the cell phenotype is present in the sample; andreceiving a second signal associated with a quantity of the plurality ofreporter molecules.
 2. The method of claim 1, wherein the reagent is adried reagent.
 3. The method of claim 1, wherein the reagent is adheredto an inner surface of the reaction chamber.
 4. The method of claim 1,wherein the portion of the cell phenotype includes a bacteria phenotypethat is resistant to an antibiotic individually or in combination withanother antibiotic.
 5. The method of claim 4, wherein the antibiotic isany one of Beta-lactams, extended-spectrum beta-lactams,Aminoglycosides, Ansamycins, Carbacephem, Carbapenems, any generation ofCephalosporins, Glycopeptides, Lincosamides, Lipopeptide, Macrolides,Monobactams, Nitrofurans, Oxazolidonones, Penicillins, Polypeptides,Quinolones, Fluoroquinolones, Sulfonamides, Tetracyclines, mycobacterialantibiotics, Chloramphenicol, Mupirocin.
 6. The method of claim 1,wherein the portion of the cell phenotype includes a bacteria phenotypethat are resistant to at least one of cefoxitin, vancomycin,teicoplainin, ampicillin/sulbactam, ciprofloxacin, meropenem,ceftazidime, ceftriaxone, piperacillin/tazobactam, or gentamicin.
 7. Themethod of claim 1, wherein: the reagent includes an antibiotic and acolorant, the antibiotic formulated to suppress production of theplurality of reporter molecules in the portion of the cell phenotype;and the first signal is associated with the colorant.
 8. The method ofclaim 1, wherein the first signal is associated with a volume of theassay media within the reaction chamber.
 9. The method of claim 1,wherein a magnitude of the second signal is independent from a quantityof the plurality of transduction particles above a predeterminedquantity.
 10. The method of claim 1, wherein the plurality oftransduction particles is engineered to be incapable of either of lyticor lysogenic replication.
 11. The method of claim 1, wherein theplurality of transduction particles is derived from a bacteriophage. 12.The method of claim 1, further comprising: disposing a substance intothe sample, the substance formulated to react with the plurality ofreporter molecules to generate the second signal.
 13. A method,comprising: receiving a container containing a swab and a transportmedia, the swab including a shaft having a collection portionconstructed from non-wound material, the transport media including asample released from the collection portion; transferring the transportmedia and the sample into a reaction chamber; mixing with the transportmedia in the reaction chamber a plurality of transduction particlesassociated with a target cell, the plurality of transduction particlesengineered to include a nucleic acid molecule formulated to cause thetarget cell to produce a plurality of reporter molecules; maintainingthe mixture of the transport media and the plurality of transductionparticles at a temperature of at least 20 C for a period of about eighthours or less to express the plurality of reporter molecules when thetarget cell is present in the sample; and receiving a signal associatedwith a quantity of the plurality of reporter molecules.
 14. The methodof claim 13, wherein the plurality of transduction particles isnon-replicative.
 15. The method of claim 13, wherein the plurality oftransduction particles is devoid of a reporter molecule of the pluralityof report molecules.
 16. The method of claim 13, wherein a reportermolecule from the plurality of reporter molecules is any one of abacterial luciferase, an eukaryotic luciferase, a fluorescent protein,an enzyme suitable for colorimetric detection, a protein suitable forimmunodetection, a peptide suitable for immunodetection or a nucleicacid that function as an aptamer or that exhibits enzymatic activity.17. The method of claim 13, wherein the maintaining is performed for aperiod of about two hours or less.
 18. The method of claim 13, whereinthe collection portion is constructed from a foam material.
 19. Themethod of claim 13, wherein: the reaction chamber contains a lyophilizedreagent tablet formulated to mix with the transport media, thelyophilized reagent tablet including an antibiotic.